Meningococcal vaccine based on lipooligosaccharide (LOS) originating from modified Neisseria meningitidis strains of immunotype L6

ABSTRACT

The invention especially relates to multivalent vaccine compositions that can treat or prevent at least 60, preferably 75% of infections caused by  Neisseria meningitidis  especially of serogroup B. To this end, the invention in particular provides a lipooligosaccharide (LQS) of  N. meningitidis  in particular constituted by a lipid A, an inner core, an α chain of L6 or L8 type, in which the heptose II residue of the inner core bears in position O-3 and in position O-6 or O-7 a phosphoethanolamine (PEA) substituent, and also to the construction of the strain of  N. meningitidis  that is capable of expressing such an LOS. The invention also relates to a strain of  N. meningitidis  of serogroup A that bears a lipooligosaccharide (LOS) in particular constituted by a lipid A, an inner core, an α chain of L6 type, in which the heptose II residue of the inner core bears in position O-3 a phosphoethanolamine (PEA) substituent and does not bear a PEA substituent in positions O-6 and O-7. The LOSs cited or originating from the mentioned strains may be used as vaccine antigens, especially in multivalent, e.g. divalent compositions, so as to offer protection against the major epidemiological complexes of  N. meningitidis , especially of scrogroup B.

This application is a continuation of U.S. application Ser. No.14/756143, filed on Aug. 5, 2015, which is a divisional of U.S.application Ser. No. 12/800454, filed on May 14, 2010, now U.S. Pat. No.9,132,181, which claims priority to U.S. Provisional Patent ApplicationSer. No. 61/271,986 filed on Jul. 29, 2009, the contents of each ofwhich are incorporated herein by reference in their entirety.

The invention relates to the field of vaccines for combating infectionscaused by Neisseria meningitidis and especially proposes a vaccinecomposition comprising two types of lipooligosaccharide (LOS), each ofthe two types originating from a particular strain of N. meningitidis.The invention also proposes intermediate means contributing toward thepreparation of the vaccine composition mentioned above.

In the field of vaccines, one of the major challenges in the near futurewill especially be the marketing of a vaccine intended for preventingall the infections caused by N. meningitidis serogroup B. This bacteriumis responsible for a certain number of pathologies, the dominant ones ofwhich are meningitis and meningococcia, but also arthritis andpericarditis. Meningococcia may be complicated with purpura fulminansand fatal septic shock.

In general, meningitis is either of viral origin or of bacterial origin.In developed countries, the bacteria mainly responsible are: N.meningitidis and Streptococcus pneumoniae, which are involved,respectively, in about 40% and 50% of the cases of bacterial meningitis.In developing countries, Haemophilus influenzae also remains asignificant source of meningitis.

In France, about 600 to 800 cases of N. meningitides-mediated meningitisare reported per year. In the United States, the number of cases risesto about 2500 to 3000 per year.

The species N. meningitidis is subdivided into serogroups according tothe nature of the capsule polysaccharides. Although there are about adozen serogroups, 90% of the cases of meningitis are attributable to theserogroups: A, B, C, Y and W135.

Efficient vaccines based on capsule polysaccharides exist for preventingthe N. meningitidis-mediated meningitis of the serogroups A, C, Y andW135. These polysaccharides per se are not or are only sparinglyimmunogenic in children under 2 years old and do not induce any immunememory. However, these drawbacks may be overcome by combining thesepolysaccharides with a carrier protein.

However, the capsule polysaccharide of N. meningitidis serogroup B isnot or only sparingly immunogenic in man, whether or not it is inconjugated form (Bruge et al, Vaccine (2004) 22: 1087). Moreover, thispolysaccharide bears an epitope that might potentially undergo a crossreaction with human tissues. Thus, it appears highly desirable to searchfor a vaccine for combating meningitis induced by N. meningitidisespecially of the serogroup B other than a vaccine based on capsulepolysaccharide.

The liposaccharide (LPS) is a major constituent of the outer membrane ofthe wall of Gram-negative bacteria. LPS is toxic at high doses tomammals and, in view of this biological activity, has been called anendotoxin. It is responsible for septic shock, a fatal pathology whichdevelops following acute infection with a Gram-negative bacterium.

Nevertheless, LPS is not only toxic, it is also immunogenic. In mammals,anti-LPS antibodies are generated during carrying and infection and canbe protective. Thus, the use of LPS has already been envisioned in theprophylaxis of infections due to Gram-negative bacteria and associateddiseases.

The structure of LPS is in particular constituted of a lipid portion,called lipid A, covalently bonded to a polysaccharide portion.

Lipid A is responsible for the toxicity of LPS. It is highly hydrophobicand enables the LPS to be anchored in the outer membrane of the wall.Lipid A is composed of a disaccharide structure substituted with fattyacid chains. The number and the composition of the fatty acid chainsvary from one species to the other.

The polysaccharide portion is in particular constituted of carbohydratechains which are responsible for the antigenicity. At least 3 majorregions can be distinguished in this polysaccharide portion:

-   (i) an inner core in particular constituted of monosaccharides [one    or more KDO (2-keto-3-deoxyoctulosonic acid) and one or more    heptoses (Hep)] which are invariant within the same bacterial    species;-   (ii) an outer core bonded to heptose and in particular constituted    of various monosaccharides; and-   (iii) an O-specific outer chain in particular constituted of a    series of repeating units—these repeating units themselves being    composed of one or more different monosaccharides.

The structure of LPS varies from one species to another. This is why,for example, in a certain number of non-enteric Gram-negative bacteriasuch as Neisseriae, Bordetellae, Branhamellas, Haemophilus andMoraxellae, the O-specific chain does not exist. The LPS saccharideportion of these bacteria is in particular constituted only of theoligosaccharide core. Consequently, the LPS from these bacteria is oftencalled lipooligosaccharide (LOS).

The structure of the LPS varies not only from one species to another,but also within the same species.

Thus, not all the strains of N. meningitidis have an LOS of the samestructure, although all the LOSs of meningococcus share the basicstructure, which is represented schematically in the followingstructural formula:

The outer core (or a chain) is variable as a function of the type ofoligosaccharide (substituent R1) attached to the glucose residue borneby the heptose I.

Whereas lipid A is essentially invariable, the inner core, which itselfalso is formed in an invariant manner from two KDO(2-keto-3-deoxyoctulosonic acid) and two heptoses (HepI and HepII),bears various substituents (i) on heptose II (substituents R2 and R3);and (ii) on the γ chain, formed from an N-acetyl glucosamine (GlcNAc),which may or may not be O-acetylated. In the literature, the R2 residueis commonly referred to as the β chain, when R2 is a glucose residue.

Originally, the LOS from N. meningitidis was listed as 13 immunotypes(IT L1 to L13), as a function of its reactivity with a series ofantibodies (Achtman et al, 1992, J. Infect. Dis. 165: 53-68). Themajority of invasive strains with N. meningitidis serogroup B is ofimmunotype L3,7 as demonstrated by the reactivity of these strains witha monoclonal antibody called L3,7,9. This monoclonal antibody is capableof recognizing each of the immunotypes L3, L7 and L9 (Gu et al, J. Clin.Microbiol. (1992) 30: 2047; Moran et al, Infect. Immun. (1994) 62 (12):5290; et Scholten et al, J. Med. Microbiol. (1994) 41: 236). Thedifferences between immunotypes originate from variations in thecomposition and in the conformation of the oligosaccharide chains. Thisshows in the table below (Table I), derived from Table 2 of Braun et al,Vaccine (2004) 22: 898, supplemented with data obtained subsequently andrelating to immunotypes L9

(Schoudhury et al, Carbohydr. Res. (2008) 343: 2771) and L11 (Mistrettaet al, (2008) Poster at the 16th International Pathogenic NeisseriaConference, Rotterdam):

TABLE I IT R1 (α chain) R2 R3 L1 NeuNAcα2-6Galα1-4Galβ1-4 PEA-3 — L2NeuNAcα2-3Galβ1-4GlcNAcβ1-3Galβ1-4 Glcα (1-3) PEA-6 or PEA-7 L3NeuNAcα2-3Galβ1-4GlcNAcβ1-3Galβ1-4 PEA-3 — L4NeuNAcα2-3Galβ1-4GlcNAcβ1-3Galβ1-4 — PEA-6 L5NeuNAcα2-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-4 Glcα (1-3) — L6GlcNAcβ1-3Galβ1-4 — PEA-6 or PEA-7 L7 Galβ1-4GlcNAcβ1-3Galβ1-4 PEA-3 —L8 Galβ1-4 PEA-3 — L9 Galβ1-4GlcNAcβ1-3Galβ1-4 — PEA-6 L10 n.d. n.d.n.d. L11 Glcβ1-4 PEA-3 PEA-6. L12 n.d n.d. n.d. L13 n.d n.d. n.d. n.d.:not determined.

It may be noted, inter alia, that certain LOSs may be sialylated(presence of N-acetylneuraminic acid on the terminal galactose residue(Gal) of the α chain). Thus, immunotypes L3 and L7 differ only by therespective presence/absence of this sialylation. Moreover, most LOSs aresubstituted with an O-acetyl group on the glucosamine residue (α-GlcNAc)of the inner core (Wakarchuk et al. (1998) Eur. J. Biochem. 254: 626;Gamian et al. (1992) J. Biol. Chem. 267: 922; Kogan et al (1997)Carbohydr. Res. 298: 191; Di Fabio et al. (1990) Can. J. Chem. 68: 1029;Michon et al. (1990) J. Biol. Chem. 275: 9716; Choudhury et al. (above);and Mistretta et al. (above)).

The other variations in the structure of the LOS are due to differentgenetic factors, including:

-   (i) the presence/absence of certain genes involved in the LOS    biosynthetic pathway and in possible mutual associations of the    genes;-   (ii) the phase variation to which certain genes are subjected;-   (iii) homologous recombination [since certain genes have conserved    regions (lgtB, lgtE and lgtH) and other genes are hybrid (lgtZ is    the hybrid of the genes lgtA and lgtB), rearrangements may take    place]; and-   (iv) mutations.

The genes involved in the LOS biosynthetic pathway (with the exceptionof two) are divided into three loci (lgt-1, lgt-2 and lgt-3). Thedescription of these genes and their function is given later,illustrated schematically by FIG. 1, which shows the structure of theLOS of N. meningitidis, the various sites at which the variability isexpressed and also the levels of intervention of the genes.

The off-locus genes are lpt3 and lot3. The gene lpt3 codes for a PEAtransferase. This enzyme has the capacity to attach aphosphoethanolamine (PEA) residue in the O-3 position of heptose II. Thegene lot3 codes for an LOS O-acetyltransferase that has the capacity toO-acetylate the γ chain. It is subject to a phase variation.

The lgt-1 locus comprises 7 genes: lgtA, lgtB, lgtC, lgtD, lgtE, lgtHand lgtZ, each coding for a particular glycosyl transferase. Among thesegenes, lgtA and lgtC are subject to a phase variation. lgtE and lgtHhave an allelic variation: the codon that determines the amino acid inposition 153 codes either for a threonine residue (and in this case theresulting enzyme is a Gal-transferase) or for a methionine residue (andin this case the resulting enzyme is a Glc-transferase).

The lgt-1 locus is classed into 8 genetic types (Zhu et al, Microbiology(2002) 148: 1833).

The lgt-2 locus comprises 2 genes: lgtF and lgtK coding forglycosylases. The product of the lgtF gene intervenes in theconstruction of the α chain by enabling the binding of the glucoseresidue to heptose I, and therefore does not intervene in the nature ofthe immunotype; nor, for that matter, does the gene lgtK.

The lgt-3 locus comprises 2 genes: lgtG and lpt6. The gene lgtG codesfor a Glc synthetase that has the capacity to attach a glucose residuein the O-3 position of heptose II. The gene lpt6 codes for a PEAtransferase that has the capacity to attach a phosphoethanolamine (PEA)substituent in the O-6 or O-7 position of heptose II. The gene lgtG issubject to a phase variation. When it is “On” and accompanied by afunctional lpt3 gene, the attachment of the glucose residue always takesplace at the expense of PEA (whose attachment is mediated by lpt3).

The lgt-3 locus is classed into 5 genetic types (Wright et al., J. Bact.(Oct. 2004) : 6970).

The Galβ1-4GlcNAcβ1-3 Galβ1-4Glcβ1-4 carbohydrate unit orlacto-N-neotetraose unit which is present in the α chain of certain N.meningitidis LOS immunotypes constitutes an epitope which canpotentially crossreact with human erythrocytes. Thus, with a view toproducing a vaccine for use in humans, it is advisable to choose an LOSwhich does not possess this unit. It may therefore be particularlyadvantageous to use an LOS originating from strains of immunotype L6 orL8.

However, a genotype study of epidemiological strains made it possible todiscover that the strains of immunotype L6 or L8 needed to be optimizedin order to modify the structure of their original LOS; this being inorder to manufacture an improved LOS-based vaccine.

The genotype study in question was performed in two stages.

First, about twenty strains of N. meningitidis were analyzed both bygenotyping and by biochemical analysis (mass spectrometry and nuclearmagnetic resonance). In a first stage, the immunotype of these strainswas predicted from the genotyping results. The results of thebiochemical analysis revealed an excellent correlation between thestructure effectively determined and the immunotype predicted bygenotyping. Given the close parallel between the genotyping results andthe biochemical analysis results, it was possible in a second stage tovalidly continue the analysis of a much broader collection of strains,solely by genotyping.

Thus, a collection of 163 strains was gathered, most of which wereprovided by the laboratories of Drs D. A. Caugant (NIPH, Oslo, Norway),D. Martin (ESR, Porirua, New Zealand) and M. Diggle (SMPRL, Glasgow,United Kingdom). The strains of this collection are sourced worldwide.About half of them were isolated in Europe. They are in the very largemajority of serogroup B and are divided into the 6 major epidemiologicalcomplexes found in Europe in the invasive strains of the serogroup B:i.e. the MLST (multilocus sequence type) ST-8, ST-11, ST-18, ST 32,ST-41/44 and ST-269 complexes.

Specifically, epidemiologically, the most recent European data indicatethat 64% of the invasive strains of meningo B are divided among these 6complexes, whereas, at the present time, 50 complexes have beendescribed (19% of the European strains not having been assigned to adetermined complex).

The table below (Table II) provides further details concerning these 163strains and indicates in parentheses the previous nomenclature or namesof the corresponding MLEE (multi electrophoretic enzyme) complexes orelectrophoretic (ET) complexes:

TABLE II Origin (number Number of of Period of Complex strainscountries) isolation Main source ST-41/44 34 9 1963-1994 D. Caugant/(lineage III) D. Martin ST-32 53 10 1981-1996 D. Caugant (ET-5) ST-26914 2 1988-2007 D. Caugant ST-18 7 2 1985-2005 D. Caugant ST-8 28 121967-1994 D. Caugant/ (cluster A4) M. Diggle ST-11 27 7 1969-1988 D.Caugant/ (ET-37) M. Diggle

The genes participating in the biosynthesis of the LPS that wereanalyzed by genotyping are the following: lgtA, lgtB, lgtC, lgtE andlgtH; lgtF; lgtG and lpt6; lpt3 and lot3. This analysis made it possibleto predict the structure of the LOS in the 6 major epidemiologicalcomplexes of meningococcus B. The results are given in FIG. 2. It willbe noted that the genotyping results for certain strains are such thatit had to be deduced that such a strain is capable of manufacturingseveral types of LOS. The LOS of such a strain may thus be classed intoseveral categories. To conclude, this explains, for example, why astrain can be compatibilized in several categories of α chain.

The results of the genotyping relating to 3 of the 4 genes involved inthe biosynthesis of the inner core and acting on its variability (i.e.lgtG, lpt6 and lpt3) and also the structure deduced therefrom arepresented in detail in Table III below:

TABLE III Combination between clonal complexes and phenotype/genotype ofthe inner core ST complexes Sum 41/44 8 of (lineage 32 (cluster 11 theDistribution of the 6 major epidemiological III) (ET-5) 269 18 A4)(ET-37) strains complexes in the invasive strains of serogroup B 25% 20%7% 4% 4% 4% 64% No lpt3− lgtG− lpt6− 6 7 No substitution lpt3+ lgtG Offlpt6− 1  4.3% substitution deleted lpt3− Glu-3 lpt3− lgtG On lpt6− 10Glu-3 lpt3+ lgtG On lpt6− 9 1  6.13% PEA-3 lpt3+ lgtG− lpt6− 33 14 92PEA-3 lpt3+ lgtG Off lpt6− 44 1 56.44% PEA-3 lpt3+ lgtG− lpt6+ 32 PEA-3PEA-6 lpt3+ lgtG Off lpt6+ 13 19 19.63% PEA-6 Glu-3 lpt3− lgtG On lpt6+21 Glu-3 PEA-6 lpt3+ lgtG On lpt6+ 13 8 12.88% PEA-6 PEA-6 lpt3− lgtG−lpt6+ 1 PEA-6 lpt3− lgtG Off lpt6+ 1  0.60% Number of strains tested 3453 14 7 28 27 163

Moreover, the genotyping study relating to the lot3 gene reveals thatthe vast majority of the strains tested O-acetylate their LOS (lot3 genepresent and “On”).

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows the structure of the LOS of N. meningitidis, the varioussites at which the variability is expressed and also the levels ofintervention of the genes.

FIG. 2 shows the results of the genotyping analysis of the lgtA, lgtB,lgtC, lgtE and lgtH; lgtF; lgtG and lpt6; lpt3 and lot3 genes thatparticipate in the biosynthesis of LPS.

FIG. 3 shows the ELISA titers expressed as log_(in) of the anti-LOS IgGsof the rabbit sera of groups A, B, C, D, E and F in the Immunogenicitystudy No. 1 in rabbits.

In coherence with the results of the genotyping study relating to thelgtG, lpt6 and lpt3 genes, a vaccine composition intended to prevent ortreat infections caused by N. meningitidis is proposed, which comprisesone or more LOSs of N. meningitidis; this being (i) so as to treat orprevent at least 70%, advantageously at least 75% and preferably atleast 80% of infections caused by N. meningitidis, especially caused byN. meningitidis serogroup B or (ii) to vaccinate against 75 to 90%,advantageously from 75 to 100%, preferably from 80 to 90% and mostpreferably particularly from 80 to 100% of infections caused by N.meningitidis, especially of serogroup B.

To this end, the choice of an LOS bearing an α chain of L6 type and aheptose II residue of the inner core substituted in the O-3 positionwith a phosphoethanolamine residue proves to be particularlyadvantageous. Now, as reported previously in Table I above, the L6immunotype strains bear only one PEA substituent in position 6/7.

Thus, when the vaccine composition comprises only one LOS, the latterhas to be necessarily optimized. When the vaccine composition comprisesat least two LOSs, one of the at least two LOSs must necessarily havebeen optimized; the second may be a natural (non-optimized) LOS.

In order to be able to manufacture a vaccine according to the invention,three strain manufacturing processes are first proposed, listed asfollows:

-   (i) a process of making a strain of N. meningitidis exhibiting a    lipooligosaccharide (LOS) in particular constituted by (comprising)    a lipid A, an inner core, an a chain of L6 type, in which the    heptose II residue of the inner core bears in position O-3 and in    position O-6 or O-7 a phosphoethanolamine (PEA) substituent; wherein    an N. meningitidis strain of immunotype L6 is modified such that it    expresses an lpt3 gene;-   (ii) a process of making an N. meningitidis strain exhibiting a    lipooligosaccharide (LOS) in particular constituted by (comprising)    a lipid A, an inner core, an a chain of L6 type, in which the    heptose II residue of the inner core bears in position O-3 a    phosphoethanolamine (PEA) substituent and does not bear a PEA    substituent in positions O-6 and O-7; wherein an N. meningitidis    strain of immunotype L6 is modified such that it expresses an lpt3    gene and such that it no longer expresses the lpt6 gene; and-   (iii) a process of making an N. meningitidis strain exhibiting a    lipooligosaccharide (LOS) in particular constituted by a lipid A, an    inner core, an α chain of L8 type, in which the heptose II residue    of the inner core bears in position O-3 and in position O-6 or O-7 a    phosphoethanolamine (PEA) substituent; wherein an N. meningitidis    strain of immunotype L8 is modified such that it expresses an lpt6    gene.

The N. meningitidis serogroup against which it is imperative to proposea vaccine in priority is the serogroup B (vaccines against the otherprevalent serogroups A, C, Y and W135 are already available).Purification of the LOS from a strain of serogroup B may lead to aproduct containing the undesirable residual B capsule in the vaccine. Toovercome these difficulties, it has now been found that an ad hoc LOSderived from strains of serogroup A can satisfy the needs in terms ofvaccination against the serogroup B. This is why, in the manufacturingprocesses (i) and (ii) according to the invention, an N. meningitidisstrain of immunotype L6, serogroup A is preferably used as startingstrain.

A strain that may be used in the manufacturing processes may express anLOS bearing a non-detoxified lipid A. In particular, the msbB gene maybe functional.

A strain of this type is the strain C708 filed on Mar. 11, 2008 at theCollection Nationale de Culture de Microorganisme, 25 rue du Dr Roux75015 Paris, according to the terms of the treaty of Budapest. Thisstrain bears the order number CNCM I-3942. This strain bears, interalia, an active lgtA gene (gene switched “ON”); an lgtBgene—(non-functional gene); an lgtG gene (switched “Off”); a truncatedlpt3 gene; an active lpt6 gene; an active lot3 gene; and an active msbBgene.

The strain C708 comprises a truncated lpt3 gene. To modify it such thatthe LOS bears a PEA substituent in position O3, the functionality of thelpt3 gene can be restored, especially by homologous recombination usinga complete (full-length) lpt3 gene. When this strain is used in process(ii) according to the invention, it is also appropriate to deactivatethe lpt6 gene so as to obtain a strain that no longer expresses thisgene. The deactivation of this gene may especially be achieved by totalor partial deletion of the lpt6 gene or alternatively by insertion of anon-pertinent sequence into the gene, for example anantibiotic-resistant gene.

According to another aspect, a subject of the invention is also:

-   (i) an N. meningitidis strain, especially of serogroup A, exhibiting    a lipooligo-saccharide (LOS) in particular constituted by a lipid A,    an inner core, an α chain of L6 type , in which the heptose II    residue of the inner core bears in position O-3 and in position O-6    or O-7 a phosphoethanolamine (PEA) substituent;-   (ii) an N. meningitidis strain of serogroup A, which exhibits a    lipooligosaccharide (LOS) in particular constituted by a lipid A, an    inner core, an α chain of L6 type, in which the heptose II residue    of the inner core bears in position O-3 a phosphoethanolamine (PEA)    substituent and does not bear a PEA substituent in positions O-6 and    O-7;-   (iii) a lipooligosaccharide (LOS) of N. meningitidis in particular    constituted by a lipid A that may or may not be detoxified, an inner    core, an α chain of L6 type, in which the heptose II residue of the    inner core bears in position O-3 and in position O-6 or O-7 a    phosphoethanolamine (PEA) substituent; and-   (iv) a lipooligosaccharide (LOS) of N. meningitidis in particular    constituted by a non-detoxified lipid A, an inner core, an α chain    of L6 type, in which the heptose II residue of the inner core bears    in position O-3 a phosphoethanolamine (PEA) substituent and does not    bear a PEA substituent in positions O-6 and O-7.

Advantageously, the strains according to the invention have a functionallot3 gene, in “On” phase variation such that the LOS that they produceis O-acetylated, at least partially, on their γ chain. The strainsaccording to the invention may bear a functional msbB gene and/orexpress an LOS in particular constituted by a non-detoxified lipid A.

Advantageously, an LOS according to the invention bears a γ chain thatis O-acetylated, at least partially.

An LOS included in the composition of a vaccine according to theinvention may be prepared according to one of the following threeprocesses:

-   (i) a process for preparing a lipooligosaccharide (LOS) of N.    meningitidis in particular constituted by a lipid A, an inner core,    an α chain of L6 type, in which the heptose II residue of the inner    core bears in position O-3 and in position O-6 or O-7 a    phosphoethanolamine (PEA) substituent; wherein (a) a strain obtained    from the manufacturing process (i) according to the invention or the    strain (i) according to the invention is cultured, and (b) the LOS    is harvested from the culture obtained in step (a);-   (ii) a process for preparing a lipooligosaccharide (LOS) of N.    meningitidis in particular constituted by a lipid A, an inner core,    an α chain of L6 type, in which the heptose II residue of the inner    core bears in position O-3 a phosphoethanolamine (PEA) substituent    and does not bear a PEA substituent in positions O-6 and O-7;    wherein (a) a strain obtained from the manufacturing process (ii) or    the strain (ii) according to the invention is cultured, and (b) the    LOS is harvested from the culture obtained in step (a); and-   (iii) a process for preparing a lipooligosaccharide (LOS) of N.    meningitidis in particular constituted by a lipid A, an inner core,    an α chain of L8 type, in which the heptose II residue of the inner    core bears in position O-3 and in position O-6 or O-7 a    phosphoethanolamine (PEA) substituent; wherein (a) a strain obtained    from the manufacturing process (iii) is cultured and (b) the LOS is    harvested from the culture obtained in step (a).

In each of the three preparation processes, the LOS may be harvestedeither in a form combined with outer membrane vesicles (OMVs), orextracted so as to be purified thereafter.

The first case amounts to harvesting OMVs that contain LOS. Such OMVsmay be isolated according to many techniques. See, for example, WO04/014417, Fredriksen et al., NIPH Annals (1991) 14: 67-79; Zollinger etal., J. Clin Invest (1979) 63: 836-848; Saunders et al., Infect. Immun.(1999) 67: 113-119; and Drabick et al., Vaccine (1999) 18: 160-172.These techniques may be divided into two major groups; those usingdeoxycholate (DOC) at a concentration of about 0.5%; and those usinglower concentrations or no DOC at all. The techniques that dispense withDOC have the advantage of concentrating the LOS in the OMVs (about10-fold more LOS than in the OMVs extracted via a technique using DOC).However, the techniques using low doses of DOC may also be advantageousin that they can reduce the residual toxicity of the LOS while at thesame time making it possible to obtain advantageous concentrations ofLOS. Once extracted, the OMVs can be purified by ultrafiltration ordiafiltration techniques known to those skilled in the art anddescribed, for example, in Frasch et al. “Outer membrane protein vesiclevaccines for meningococcal disease” in Methods in Molecular Medicine,vol. 66, Meningococcal Vaccines: Methods and Protocols (2001): 81-107(Edited by A J. Pollard and M. C. Maiden, Humana Press Totowa, N.J.).

In the second case, the LOS is extracted from a bacterial culture andthen purified according to standard methods. Many purification processesare described in the literature. By way of example, mention is made ofGu & Tsai, 1993, Infect. Immun. 61 (5): 1873, Wu et al., 1987, Anal.Biochem.160: 281 and U.S. Pat. No. 6,531,131. An LPS preparation canalso be quantified by using known techniques. A practical methodconsists in assaying the KDO by HPAEC-PAD chromatography (highperformance anion exchange chromatography).

Detoxification of the LOS

For incorporation into a vaccine, the LOS needs to be detoxified. Thetoxicity of the LOS is due to its lipid A. However, it is neitherimperative to remove the lipid A in its entirety; nor to modify it, forexample by mutation (e.g. msbB minus mutation). In fact, since thetoxicity is more particularly linked to a supramolecular conformationconferred by all the fatty acid chains borne by the disaccharide nucleusof the lipid, according to one advantageous embodiment, it is sufficientto act on these chains.

The detoxification can be obtained according to various approaches:chemical, enzymatic or genetic or alternatively by complexation with apeptide analog of polymyxin B or alternatively byincorporation/formulation into liposomes.

The level of detoxification of the LOS can be assessed inter aliaaccording to one of the two following standard tests:

-   -   the pyrogenic test in rabbits. This test, the calculations and        the reading thereof have been implemented according to the        principles set out in the European Pharmacopoeia (Edition 6.0,        paragraph 2.6.8.).    -   the LAL test (Limulus Amebocyte Lysate) implemented according to        the principles set out in the European Pharmacopoeia (Edition        6.0, paragraph 2.6.14.).

Detoxification via the Chemical Route

The chemical approach consists in treating the LOS with a chemicalagent. According to one particular embodiment, the LOS is subjected tomild acid hydrolysis with acetic acid which removes the lipid A and alsothe branched KDO(s) when it (they) is (are) present in the LOSstructure. Such a treatment is, for example, described in Gu & TsaiInfect. Immun. (1993) 61: 1873. According to an alternative andpreferred embodiment, the LOS is subjected to a de-O-acylation,preferably a primary de-O-acylation, i.a. by treatment with hydrazine,which hydrolyzes the esterified primary fatty acid chains of the lipidA. Such a treatment is, for example, described in U.S. Pat. No.6,531,131, Gupta et al, Infect. Immun. (1992) 60 (8): 3201 and Gu et al,Infect. Immun. (1996) 64 (10): 4047.

Detoxification via the Enzymatic Route

The enzymatic approach consists in placing the LOS in the presence oflipases capable of digesting the esterified fatty acid chains of thelipid A. Such lipases are produced by the amoeba Dictyosteliumdiscoideum. According to one particularly advantageous embodiment, theamoeba and a Gram-negative bacterium that can be phagocytosed by theamoeba, such as N. meningitidis, are cultured together (coculture). Thesupernatant is then recovered and the LOS is extracted from thesupernatant which is then free of fatty acid chains. It may also be anacyloxyacyl hydrolase produced by certain human cells (patent WO87/07297 Munford R.) or by Salmonella typhimurium (Trent et al 2001 J.Biol. Chem. 276: 9083-9092; Reynolds et al. 2006 J. Biol. Chem. 281:21974-21987) (enzyme encoded by the PagL or LpxR genes in the lattercase).

Detoxification via the Genetic Route

The genetic approach consists in using an LOS produced by a bacterialstrain of which the genotype is such that the entity of the LOS normallyresponsible for its toxicity (lipid A, and more particularly the lipidtails of lipid A) has a greatly reduced or even nonexistent degree oftoxicity. Such a bacterial strain can be conveniently obtained bymutation. Starting from a wild-type strain (i.e. a strain producing atoxic LOS), this then involves inactivating, by mutation, certain genesinvolved in the biosynthesis of the fatty acid chains, or in theattachment thereof to the disaccharide nucleus of lipid A. Thus, it ispossible to envision inactivating the ipxL1 or ipxL2 genes (also calledhtrB1/htrB2) of N. meningitidis or equivalents thereof in other species(for example, the equivalents of the meningococcal ipxL1 and ipxL2 genesare respectively called msbB or ipxM and htrB or ipxL in E. coli.). Amutation that inactivates one of these genes results in an LOS devoid ofone or of two secondary acyl chains. ipxL1 or L2 mutants of N.meningitidis or of Haemophilus influenzae are in particular described inpatent applications WO 00/26384, U.S. 2004/0171133 and WO 97/019688. InN. meningitidis, the endogenous ipxA gene can also be replaced with thehomologous gene originating from E. coli or Pseudomonas aeruginosa. Thefatty acid chains thereof are modified, resulting in a less toxic lipidA (Steeghs et al, Cell. Microbiol. (2002) 4 (9): 599). The geneticapproach is favored when the LOS is purified in the form of OMVs.

LOS Detoxified by Complexation with a Peptide Analog of Polymyxin B

A fourth approach consists in complexing the LOS with a peptide analogof polymyxin B, as is, for example, described in patent application WO06/108586. The LOS that is complexed and consequently detoxified iscalled endotoxoid.

The polymyxin B analog included in the composition of an endotoxoid thatis useful for the purposes of the present invention may be any peptidethat is capable of detoxifying the LPS by simple complexation. Suchpeptides are especially described in patent or patent applications U.S.Pat. No. 6,951,652, EP 976 402 and WO 06/108 586.

Thus, an advantageous peptide may be the peptide of formula (I)

NH₂-A-Cys1-B-Cys2-C-COOH (SEQ ID NO: 1), in which:

A is a peptide of 2 to 5 and preferably 3 or 4 amino acid residues, inwhich at least 2 amino acid residues are independently chosen from Lys,Hyl (hydroxylysine), Arg and His;B is a peptide of 3 to 7 and preferably 4 or 5 amino acid residues,which comprises at least two and preferably three amino acid residueschosen from Val, Leu, Ile, Phe, Tyr and Trp; andC is optional (this position may or may not be empty) and is an aminoacid residue or a peptide formed from 2 to 3 amino acid residues;on condition that the cationic amino acid/hydrophobic amino acid ratioin the peptide of formula I is from 0.4 to 2, advantageously from 0.5 to1.2 or 1.5, preferably from 0.6 to 1; better still from 0.6 to 0.8; forexample 0.75.

Preferably, in the peptide of formula (I), position C is an emptyposition.

Particular examples of the peptide of formula (I) are the followingpeptides:

(SEQ ID NO: 2) NH₂-Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Lys-Lys-Cys2-COOH(peptide SAEP2); (SEQ ID NO: 3)NH₂-Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH (peptide SAEP2-L2);.(SEQ ID NO: 4) NH₂-Lys-Arg-His-Hyl-Cys1-Lys-Arg-Ile-Val-Leu-Cys2- COOH;(SEQ ID NO: 5) NH₂-Lys-Arg-His-Cys1-Val-Leu-Ile-Trp-Tyr-Phe-Cys2- COOH;(SEQ ID NO: 6) NH₂-Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2- COOH; and(SEQ ID NO: 7) NH₂-Hyl-Arg-His-Lys-Cys1-Phe-Tyr-Trp-Val-Ile-Leu-Cys2-COOH.

The peptides of formula (I) may be in monomer form or, preferably, inparallel or antiparallel dimer form.

In general, use may also be made of a dimeric peptide of formula (II)

in which the two Cys1 residues are linked together via a disulfidebridge and the two Cys2 residues are linked together via a disulfidebridge; or of formula (III)

in which the Cys1 residues are linked to the Cys2 residues via peptideinter-chain disulfide bridges;in which formulae (II) and (III):

-   -   A and A′ are, independently, a peptide of 2 to 5 and preferably        3 or 4 amino acid residues, in which at least 2 amino acid        residues are independently chosen from Lys, Hyl (hydroxylysine),        Arg and His;    -   B and B′ are, independently, a peptide of 3 to 7 and preferably        4 or 5 amino acid residues, which comprise at least two and        preferably three amino acid residues independently chosen from        Val, Leu, Ile, Phe, Tyr and Trp; and    -   C and C′ are optional (these positions may or may not be empty)        and are, independently, an amino acid residue or a peptide of 2        to 3 amino acid residues; on condition that the cationic amino        acid/hydrophobic amino acid ratio in the dimer of formula (II)        or (III) is from 0.4 to 2, advantageously from 0.5 to 1.2 or        1.5, preferably from 0.6 to 1 and better still from 0.6 to 0.8;        for example. 0.75.

Advantageously, A and A′ are, independently, a peptide of 2 to 5 andpreferably 3 or 4 amino acid residues, in which at least one andpreferably 2 amino acid residues are independently chosen from Lys, Hyl,Arg and His; and, where appropriate, those that are not chosen from Lys,Hyl, Arg and His (“the remaining amino acid residues”) being chosen fromthe group of uncharged, polar or nonpolar amino acid residues;preferably Thr, Ser and Gly; most particularly preferably Thr.

When A and A′ comprise 3 amino acid residues, each of them may be acationic residue; or alternatively, two of the three residues arecationic amino acids, whereas the remaining residue is chosen from thegroup of uncharged, polar or nonpolar amino acid residues; preferablyThr, Ser and Gly; most particularly preferably Thr.

When A and A′ comprise 4 amino acid residues, it is preferable for twoor three of the four residues to be chosen from the groups of cationicamino acid residues as defined above, whereas the remaining residue(s)is(are) chosen from the group of uncharged, polar or nonpolar amino acidresidues as defined above.

When A and A′ comprise 5 amino acid residues, it is preferred for threeor four of the five residues to be chosen from the groups of cationicamino acid residues as defined above, whereas the remaining residue(s)is(are) chosen from the group of uncharged, polar or nonpolar amino acidresidues as defined above.

Advantageously, B and B′ are, independently, a peptide of 3 to 7 andpreferably 4 or 5 amino acid residues, which comprises at least two andpreferably three amino acid residues independently chosen from Val, Leu,Ile, Phe, Tyr and Trp; preferably Leu, Ile and Phe; and, whereappropriate, those that are not chosen from Val, Leu, Ile, Phe, Tyr andTrp (“the remaining amino acid residues”) being chosen independentlyfrom the group formed by Lys, Hyl, Arg and His. As may readily beunderstood, B and B′ may comprise up to 7 amino acid residuesindependently chosen from Val, Leu, Ile, Phe, Tyr and Trp.

Advantageously, B and B′ comprise the sequence—X1-X2-X3 —, in which X1and X2; X2 and X3; or X1, X2 and X3 are independently chosen from Val,Leu, Ile, Phe, Tyr and Trp; preferably from Leu, Ile and Phe. In onepreferred embodiment, the sequence—X1-X2-X3—comprises the Phe-Leu unit.

The particular embodiments of B and B′ include:

-   (i) the sequence —X1-X2-X3—in which:-   X1 is Lys, Hyl, His or Arg, preferably Lys or Arg; preferably Lys;-   X2 is Phe, Leu, Ile, Tyr, Trp or Val; preferably Phe or Leu; more    particularly preferably Phe; and-   X3 is Phe, Leu, Ile, Tyr, Trp or Val; preferably Phe or Leu; more    particularly preferably Leu; and-   (ii) where appropriate, the amino acid residues are each    independently chosen from the group formed by Val, Leu, Ile, Phe,    Tyr, Trp, Lys, Hyl, Arg and His; preferably Val, Leu, Ile, Phe, Tyr    and Trp; more particularly preferably Leu, Ile and Phe.

When B and B′ comprise more than 4 nonpolar amino acid residues, A andA′ preferably comprise at least 3 positively charged amino acidresidues.

In C and C′, the amino acid residues may be any amino acid residue, oncondition that the cationic amino acid residues/hydrophobic amino acidresidues ratio remains in the indicated range. Advantageously, they areindependently chosen from uncharged, polar or nonpolar amino acidresidues, the latter being preferred. However, preferably, the positionsC and C′ are empty positions.

Consequently, a preferred class of the dimers is of formula (IV)

in which the two Cys1 residues are linked together via a disulfidebridge and the two Cys2 residues are linked together via a disulfidebridge;or of formula (V)

in which the Cys1 residues are linked to the Cys2 residues via peptideinter-chain disulfide bridges;in which formulae (IV) and (V), in which A, A′, B and B′ are asdescribed above; on condition that the cationic amino acid/hydrophobicamino acid ratio in the dimer of formula (IV) or (V), is from 0.4 to 2,advantageously from 0.5 to 1.2 or 1.5, preferably from 0.6 to 1 andbetter still from 0.6 to 0.8; for example 0.75.

In formulae (II) to (V), A and A′ are preferably identical. This islikewise the case as regards B and B′, on the one hand, and C and C′, onthe other hand. A dimer of formula (II) to (V), in which A and A′; B andB′; and C and C′ are identical in pairs, is designated as a homologousdimer.

For these dimers, mention may be made, for example, of the parallel andantiparallel dimers formed from the peptide SAEP2-L2 (SEQ ID NO: 3):

The endotoxoid that is useful for the purposes of the present inventionmay advantageously be characterized by an LOS/peptide mole ratio from1/1.5 to 1/0.5, preferably from 1/1.2 to 1/0.8, and most particularlypreferably from 1/1.1 to 1/0.9, e.g.

1/1.

LOS in Detoxified Liposomes

When the LOS is formulated in liposomes, it does not appear to benecessarily required to detoxify it beforehand. This is because LOS inliposomes—i.e. associated with the lipid bilayer forming theliposomes—may experience a very substantial decrease in toxicity. Thesize of this decrease, which can be as much as a substantial loss,depends partly on the nature of the components forming the liposome.Thus, when positively charged components (components of cationic nature)are used, the loss of toxicity may be greater than with uncharged(neutral) or anionic components.

The term “liposomes” is intended to mean a synthetic entity, preferablya synthetic vesicle, formed of at least one lipid bilayer membrane (ormatrix) enclosing an aqueous compartment. For the purposes of thepresent invention, the liposomes may be uni-lamellar (a single bilayermembrane) or multi-lamellar (several membranes layered like an onion).The lipids constituting the bilayer membrane comprise a non-polar regionwhich, typically, is made of chain(s) of fatty acids or of cholesterol,and a polar region, typically made of a phosphate group and/or oftertiary or quaternary ammonium salts. Depending on its composition, thepolar region may, in particular at physiological pH (pH≈7) carry eithera negative (anionic lipid) or positive (cationic lipid) net (overall)surface charge, or not carry a net charge (neutral lipid).

For the purposes of detoxifying the LOS, the liposomes may be liposomesof any type; in particular, they may be in particular constituted of anylipid known to be of use in the production of liposomes. The lipid(s)that go(es) to make up the composition of the liposomes may be neutral,anionic or cationic lipid(s); the latter being preferred. These lipidsmay be of natural origin (plant or egg extraction products, for example)or synthetic origin; the latter being preferred. The liposomes may alsobe in particular constituted of a mixture of these lipids; for example,of a cationic or anionic lipid and of a neutral lipid, as a mixture. Inthe latter two cases, the neutral lipid is often referred to asco-lipid. According to one advantageous mixture embodiment, the charged(cationic or anionic) lipid: neutral lipid mole ratio is between 10:1and 1:10, advantageously between 4:1 and 1:4, preferably between 3:1 and1:3, limits included.

With regard to the neutral lipids, mention is made, by way of example,of: (i) cholesterol; (ii) phosphatidylcholines such as, for example,1,2-diacyl-sn-glycero-3-phosphocholines, e.g.1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and also1-acyl-2-acyl-sn-glycero-3-phosphocholines of which the acyl chains aredifferent than one another (mixed acyl chains); and (iii)phosphatidylethanolamines such as, for example,1,2-diacyl-sn-glycero-3-phosphoethanolamines, e.g.1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and also1-acyl-2-acyl-sn-glycero-3-phospho-ethanolamines bearing mixed acylchains.

With regard to the anionic lipids, mention is made, by way of example,of: (i) cholesteryl hemi succinate (CHEMS); (ii) phosphatidylserinessuch as 1,2-diacyl-sn-glycero-3-[phospho-L-serine]s, e.g.1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOP 5), and1-acyl-2-acyl-sn-glycero-3-[phospho-L-serine]s bearing mixed acylchains; (iii) phosphatidylglycerols such as1,2-diacyl-sn-glycero-3-[phospho-rac-(1-glycerol)]s, e.g.1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG), and1-acyl-2-acyl-sn-glycero-3-[phospho-rac-(1-glycerol)]s bearing mixedacyl chains; (iv) phosphatidic acids such as1,2-diacyl-sn-glycero-3-phosphates, e.g.1,2-dioleoyl-sn-glycero-3-phosphate (DOPA), and1-acyl-2-acyl-sn-glycero-3-phosphates bearing mixed acyl chains; and (v)phosphatidylinositols such as1,2-diacyl-sn-glycero-3-(phosphoinositol)s, e.g.1,2-dioleoyl-sn-glycero-3-(phosphoinositol) (DOPI), and1-acyl-2-acyl-sn-glycero-3-(phosphoinositol)s bearing mixed acyl chains.

With regard to the cationic lipids, mention is made, by way of example,of:

-   (i) lipophilic amines or alkylamines such as, for example,    dimethyldioctadecylammonium (DDA), trimethyldioctadecylammonium    (DTA) or structural homologs of DDA and of DTA [these alkylamines    are advantageously used in the form of a salt; mention is made, for    example, of dimethyldioctadecylammonium bromide (DDAB)];-   (ii)    octadecenoyloxy(ethyl-2-heptadecenyl-3-hydroxyethyl)imidazolinium    (DOTIM) and structural homologs thereof;-   (iii) lipospermines such as    N-palmitoyl-D-erythrosphingosyl-1-O-carbamoylspermine (CCS) and    dioctadecylamidoglycylspermine (DOGS, transfectam);-   (iv) lipids incorporating an ethylphosphocholine structure, such as    cationic derivatives of phospholipids, in particular phosphoric    ester derivatives of phosphatidylcholine, for example those    described in patent application WO 05/049080 and including, in    particular:    -   1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine,    -   1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine,    -   1,2-palmitoyloleoyl-sn-glycero-3-ethylphosphocholine,    -   1,2-distearoyl-sn-glycero-3-ethylphosphocholine (DSPC),    -   1,2-dioleyl-sn-glycero-3-ethylphosphocholine (DOEPC or EDOPC or        ethyl-DOPC or ethyl PC),    -   and also structural homologs thereof;-   (v) lipids incorporating a trimethylammonium structure, such as    N-(1-[2,3-dioleyloxy]propyl)-N,N,N-trimethylammonium (DOTMA) and    structural homologs thereof and those incorporating a    trimethylammonium propane structure, such as    1,2-dioleyl-3-trimethylammonium propane (DOTAP) and structural    homologs thereof; and also lipids incorporating a dimethylammonium    structure, such as 1,2-dioleyl-3-dimethylammonium propane (DODAP)    and structural homologs thereof; and-   (vi) cationic derivatives of cholesterol, such as    3β-[N′,N′-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) or    other cationic derivatives of cholesterol, such as those described    in U.S. Pat. No. 5,283,185, and in particular    cholesteryl-3β-carboxamidoethylenetrimethylammonium iodide,    cholesteryl-3β-carboxyamidoethylene-amine,    cholesteryl-3β-oxysuccinamidoethylenetrimethylammonium iodide and    3β-[N-(polyethyleneimine)carbamoyl] cholesterol.

The term “structural homologs” signifies lipids which have thecharacteristic structure of the reference lipid while at the same timediffering therefrom by virtue of secondary modifications, especially inthe nonpolar region, in particular of the number of carbon atoms and ofdouble bonds in the fatty acid chains.

These fatty acids, which are also found in the neutral and anionicphospholipids, are, for example, dodecanoic or lauric acid (C12:0),tetradecanoic or myristic acid (C14:0), hexadecanoic or palmitic acid(C16:0), cis-9-hexadecanoic or palmitoleic acid (C16:1), octadecanoic orstearic acid (C18:0), cis-9-octadecanoic or oleic acid (C18:1),cis,cis-9,12-octadecadienoic or linoleic acid (C18:2),cis-cis-6,9-octadecadienoic acid (C18:2),all-cis-9,12,15-octadecatrienoic or α-linolenic acid (C18:3),all-cis-6,9,12-octadecatrienoic or γ-linolenic acid (C18:3), eicosanoicor arachidic acid (C20:0), cis-9-eicosenoic or gadoleic acid (C20:1),all-cis-8,11,14-eicosatrienoic acid (C20:3),all-cis-5,8,11,14-eicosatetraenoic or arachidonic acid (C20:4),all-cis-5,8,11,14,17-eicosapentaneoic acid (C20:5), docosanoic orbehenic acid (C22:0), all-cis-7,10,13,16,19-docosapentaenoic acid(C22:5), all-cis-4,7,10,13,16,19-docosahexaenoic acid (C22:6) andtetracosanoic or lignoceric acid (C24:0).

According to one particular embodiment, a mixture of cationic lipid andneutral lipid is used. By way of example, mention is made of:

-   -   a mixture of DC-chol and DOPE, in particular in a DC-chol:DOPE        mole ratio ranging from 10:1 to 1:10, advantageously from 4:1 to        1:4, preferably from approximately 3:1 to 1:3;    -   a mixture of ethyl-DOPC and cholesterol, in particular in an        ethyl-DOPC:cholesterol mole ratio ranging from 10:1 to 1:10,        advantageously from 4:1 to 1:4, preferably from approximately        3:1 to 1:3; and    -   a mixture of ethyl-DOPC and DOPE, in particular in an        ethyl-DOPC:DOPE mole ratio ranging from 10:1 to 1:10,        advantageously from 4:1 to 1:4, preferably from approximately        3:1 to 1:3.

According to one advantageous method of preparation, in an initial step,a dry lipid film is prepared with all the compounds that go to make upthe composition of the liposomes. The lipid film is then rein particularconstituted in an aqueous medium, in the presence of LOS, for example ina lipid:LOS mole ratio of 100 to 500, advantageously of 100 to 400,preferably of 200 to 300, most particularly preferably of approximately250. In general, it is considered that this same mole ratio should notsubstantially vary at the end of the method of preparing the LOSliposomes.

In general, the reconstitution step in an aqueous medium results in thespontaneous formation of multilamellar vesicles, the size of which issubsequently homogenized by gradually decreasing the number of lamellaeby extrusion, for example using an extruder, by passing the lipidsuspension, under a nitrogen pressure, through polycarbonate membraneshaving decreasing pore diameters (0.8, 0.4, 0.2 μm). The extrusionprocess can also be replaced with another process using a detergent(surfactant) which disperses lipids. This detergent is subsequentlyremoved by dialysis or by adsorption onto porous polystyrene microbeadswith a particular affinity for detergent (BioBeads). When the surfactantis removed from the lipid dispersion, the lipids reorganize in a doublelayer.

At the end of the incorporation of the LOS into liposomes, a mixture inparticular constituted of ad hoc liposomes and of LOS in free form maycommonly be obtained. Advantageously, the liposomes are then purified inorder to be rid of the non-detoxified LPS in free form.

Conjugation of LOS

In a vaccine according to the invention, the LOS is advantageously inthe form of a LOS/polypeptide carrier conjugate, in particular when itis not in the form of OMVs or liposomes.

The carrier polypeptide can be any carrier polypeptide, oligopeptide orprotein in use in the conjugated vaccines field; and in particularpertussis, diphtheria or tetanus toxoid, the diphtheria toxin mutantnamed CRM197, a bacterial OMP or a bacterial protein complex (forexample, N. meningitidis OMPC (outer-membrane protein Complex)),Pseudomonas exotoxin A, Haemophilus influenzae lipoprotein D,Streptococcus pneumoniae pneumolysine, Bordetella pertussis filamentoushemagglutinin and the subunit B of the human transferrin receptor of N.meningitidis (TbpB).

Many methods of conjugation exist in the technical field. Some arelisted, for example, in patent applications EP 941 738 and WO 98/31393.

In general, the reactive groups of the LOS involved in the conjugationare those of the inner core or of lipid A. It may involve, inter alia,the acid function of the KDO, or else an aldehyde generated subsequentto an appropriate treatment on the disaccharide of lipid A. For example,a phosphatase treatment generates an aldehyde on the structure of thesecond glucosamine of lipid A from N. meningitidis (Brade H. (2002) J.Endotoxin Res. 8 (4): 295 Mieszala et al, (2003) Carbohydrate Res. 338:167 and Cox et al, (2005) Vaccine 23 (5): 5054).

Advantageously, the method of conjugation makes use (i) of abifunctional linking agent (linker) or (ii) of a spacer and of a linker.

For example, in the first case, the LOS is activated with a bifunctionalcoupling agent (linker) of formula R1-A-R2, such that the R2 radicalreacts with a reactive group of the KDO or of the lipid A in order toobtain an activated LOS; the activated LOS is then conjugated with thepolypeptide such that the R1 substituent reacts with a functional groupborne by the polypeptide, in order to obtain a conjugate.

For example, in the second case, the LOS is derivatized with a spacer offormula R3-B-R4 such that the R3 radical reacts with a reactive group ofthe KDO or of the lipid A in order to obtain a derivatized LOS; thederivatized LOS is then activated with a bifunctional coupling agent(linker) of formula R1-A-R2 such that the R2 radical reacts with the R4radical in order to obtain a derivatized and activated LOS; finally, thederivatized and activated LOS is conjugated with the polypeptide suchthat the R1 radical reacts with a functional group borne by thepolypeptide in order to obtain a conjugate.

In the second case, the process can also be carried out in the followingway: the protein is derivatized with a spacer of formula R3-B-R4 suchthat the R4 radical reacts with a functional group borne by thepolypeptide; the LOS is activated with a bifunctional linker of formulaR1-A-R2 such that the R2 radical reacts with a reactive group of the KDOor of the lipid A, in order to obtain an activated LOS; and then theactivated LOS is conjugated with the derivatized protein such that theR1 radical of the activated LOS reacts with the R3 radical of thederivatized polypeptide, in order to obtain a conjugate.

In the formula of the spacer, B may be a carbon chain, preferablycarbonyl, alkyl or alkylene, for example C1 to C12. R3 and R4 mayindependently be a thiol or amine group or a residue bearing same, forexample a hydrazide group, i.e. NH₂—NH—CO—. Compounds that may be usedas a spacer have, for example, the formula NH₂—B—NH₂, or preferablyNH₂—B—SH and NH₂—B—S—S—B′—NH₂. By way of particular example, mention ismade of: cysteamine, cysteine, diamines, e.g. diaminohexane, adipic aciddihydrazide (ADH), urea and cystamine.

In the formula of the linker, A may be an aromatic or preferablyaliphatic chain which is substituted or unsubstituted and whichadvantageously contains from 1 to 12 carbon atoms, preferably 3 to 8carbon atoms. For example, A may be a C2 to C8 alkylene, a phenylene, aC7 to C12 aralkylene, a C2 to C8 alkyl, a phenyl, a C7 to C12 aralkyl, aC6 alkanoyloxy or a benzylcarbonyloxy, which may be substituted orunsubstituted.

The R2 radical is the functional group of the linker which creates thelink with the LOS or with the derivatized LOS. Thus, R2 is a functionalgroup which can react with a carboxyl, hydroxyl, aldehyde or aminegroup. If the linker must react with a hydroxyl, carboxyl or aldehydegroup, R2 is preferably an amine group or a residue carrying an aminegroup, for example a hydrazide group, i.e. NH₂—NH—CO—. If the linkermust react with an amine group, R2 is preferably a carboxyl,succinimidyl (e.g. N-hydroxy-succinimidyl) or sulfosuccinimidyl (e.g.N-hydroxysulfosuccinimidyl) group.

Thus, compounds that can be used as a linker may be chosen from adipicacid dihydrazide (ADH); sulfosuccinimidyl6-(3[2-pyridyldithio]propionamido)hexanoate (Sulfo-LC-SPDP);succinimidyl 6-(3-[2-pyridyldithio]propionamido)hexanoate (LC-SPDP);N-succinimidyl-S-acetyl thioacetate (SATA); N-succinimidyl3-(2-pyridyldithio)propionate (SPDP), succinimidyl acetylthiopropionate(SATP); succinimidyl-4-(N-maleimidomthyl)cyclohexane-1-carboxylate(SMCC); maleimidobenzoyl-N-hydroxysuccinimide ester (MB S);N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB); succinimidyl4-(p-maleimidophenyl)butyrate (SMPB); bromoaceticacid-N-hydroxysuccinimide (BANS) ester;dithiobis-(succinimidylpropionate) (DTSSP);H-(γ-maleimidobutyryloxy)succinimide ester (GMB S);succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate;N-succinimidyl-4-(4-maleimidophenyl)butyrate; N-[β-maleimidocaproicacid] hydrazide (BMCH); N-succinimidyl-4-maleimidobutyrate; andN-succinimidyl-3-maleiimidobenzoate.

By way of example, it is proposed to use the acid function of the KDO inorder to derivatize the LOS with ADH in the presence of a carbodiimide[e.g. 3-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC)].The amine function thus introduced is then reacted with the carboxylfunctions of the polypeptide, in the presence of EDAC, after havingprotected the amine functions of the latter (Wu et al (2005) Vaccine 23:5177) or having converted them to acid functions (succinylation of theprotein; Pavliakova et al, Infect. Immun. (1999) 67 (10): 5526).

Alternatively, it is proposed to use the acid function of the KDO inorder to derivatize the LOS with cysteamine or cysteine in the presenceof EDAC. The thiol function thus introduced is then reacted with themaleimide function of a homobifunctional linker, such asbismaleimidohexane; or a heterobifunctional linker, such as GMBS. In thefirst case, the maleimide function thus introduced is then reacted withthe thiol functions of the polypeptide. In the second case, thesuccinimidyl function of the derivatized and activated LOS is reactedwith the amine functions of the polypeptide.

Depending on the method of conjugation selected, the LOS and thepolypeptide can be conjugated to one another in an LOS:polypeptide moleratio of from 10⁻¹ to 10², advantageously from 1 to 10², preferably from1 to 50; most particularly preferably of approximately 20.

As stated previously, a subject of the invention is a pharmaceuticalcomposition for combating infections caused by N. meningitidis,especially of serogroup B, which comprises:

-   (i) an N. meningitidis LOS, in particular constituted by a    non-detoxified lipid A, an inner core, an α chain of L6 type, in    which the heptose II residue of the inner core bears in position O-3    a phosphoethanolamine (PEA) substituent and does not bear a PEA    substituent in positions O-6 and O-7 or an LOS obtained according to    the preparation process (ii); and, optionally,-   (ii) an N. meningitidis LOS in particular constituted by a lipid A,    an inner core, an a chain of L6 type, in which the heptose II    residue of the inner core bears in position O-3 and in position O-6    or O-7 a phosphoethanolamine (PEA) substituent or an LOS obtained    according to the preparation process (i); and, optionally,-   (iii) an N. meningitidis LOS in particular constituted by a lipid A,    an inner core, an a chain of L6 or L8 type, in which the heptose II    residue of the inner core bears in position O-6 or O-7 a    phosphoethanolamine (PEA) substituent and does not bear a PEA    substituent in position O-3.

A subject of the invention is also a pharmaceutical composition forcombating infections caused by N. meningitidis, especially of serogroupB, which comprises an N. meningitidis LOS in particular constituted by alipid A, an inner core, an α chain of L6 type, in which the heptose IIresidue of the inner core bears in position O-3 and in position O-6 orO-7 a phosphoethanolamine (PEA) substituent; or obtained according topreparation process (i).

As indicated previously, the latter composition may also comprise an LOSobtained according to preparation process (ii) or an N. meningitidis LOSin particular constituted by a lipid A, an inner core, an α chain of L6type, in which the heptose II residue of the inner core bears inposition O-3 a phosphoethanolamine (PEA) substituent and does not bear aPEA substituent in positions O-6 and O-7; and also an N. meningitidisLOS in particular constituted by a lipid A, an inner core, an α chain ofL6 type, in which the heptose II residue of the inner core bears inposition O-6 or O-7 a phosphoethanolamine (PEA) substituent and does notbear a PEA substituent in position O-3.

A subject of the invention is also a pharmaceutical composition forcombating infections caused by N. meningitidis, especially of serogroupB, which comprises:

-   (i) an LOS of N. meningitidis, preferably of serogroup A, in    particular constituted by a lipid A, an inner core, an α chain of L8    type, in which the heptose II residue of the inner core bears in    position O-3 a phosphoethanolamine (PEA) substituent and does not    bear a PEA substituent in positions O-6 and O-7; and-   (ii) an N. meningitidis LOS, in particular constituted by a    non-detoxified lipid A, an inner core, an α chain of L6 type, in    which the heptose II residue of the inner core bears in position O-3    a phosphoethanolamine (PEA) substituent and does not bear a PEA    substituent in positions O-6 and O-7 or an LOS obtained according to    preparation process (ii); or an N. meningitidis LOS in particular    constituted by a lipid A, an inner core, an α chain of L6 type, in    which the heptose II residue of the inner core bears in position O-3    and in position O-6 or O-7 a phosphoethanolamine (PEA) substituent;    or obtained according to preparation process (i).

In the latter composition, the LOS of point (i) may advantageously bethe LOS of a strain of immunotype L8.

The invention also relates to a pharmaceutical composition againstinfections caused by N. meningitidis, especially of serogroup B, whichcomprises:

-   (i) an LOS of N. meningitidis, preferably of serogroup A, in    particular constituted by a lipid A, an inner core, an α chain of L8    type, in which the heptose II residue of the inner core bears in    position O-3 and in position O-6 or O-7 a phosphoethanolamine (PEA)    substituent; and-   (ii) an N. meningitidis LOS, in particular constituted by a    non-detoxified lipid A, an inner core, an α chain of L6 type, in    which the heptose II residue of the inner core bears in position O-3    a phosphoethanolamine (PEA) substituent and does not bear a PEA    substituent in positions O-6 and O-7 or an LOS obtained according to    preparation process (ii); or an N. meningitidis LOS in particular    constituted by a lipid A, an inner core, an α chain of L6 type, in    which the heptose II residue of the inner core bears in position O-3    and in position O-6 or O-7 a phosphoethanolamine (PEA) substituent;    or obtained according to preparation process (i).

In the latter composition, the LOS of point (i) may advantageously bethe LOS of the strain C708 lpt3FL lpt6TR lgtA::erm or the LOS of thestrain A1 (Zhu, Klutch & Tsai, FEMS Microbiology Letters (2001) 203: 173and Gu, Tsai & Karpas, J. Clin. Microbiol. (Aug 1992) 30 (8): 2047).

Thus, a divalent vaccine composition i.a. according to the invention maybe made up in various ways. The following 6 examples are mentioned:

-   1) A vaccine composition according to the invention, which    comprises:    -   (i) an LOS obtained according to preparation process (i) or        an N. meningitidis LOS in particular constituted by a lipid A,        an inner core, an α chain of L6 type, in which the heptose II        residue of the inner core bears in position O-3 and in position        O-6 or O-7 a phosphoethanolamine (PEA) substituent; and-   (ii) an LOS obtained according to preparation process (ii) or an N.    meningitidis LOS in particular constituted by a lipid A, an inner    core, an a chain of L6 type, in which the heptose II residue of the    inner core bears in position O-3 a phosphoethanolamine (PEA)    substituent and does not bear a PEA substituent in positions O-6 and    O-7,-   2) A vaccine composition according to the invention, which    comprises:    -   (i) an LOS obtained according to preparation process (i) or        an N. meningitidis LOS in particular constituted by a lipid A,        an inner core, an a chain of L6 type, in which the heptose II        residue of the inner core bears in position O-3 a        phosphoethanolamine (PEA) substituent and does not bear a PEA        substituent in positions O-6 and O-7; and    -   (ii) an N. meningitidis LOS in particular constituted by a lipid        A, an inner core, an α chain of L6 type, in which the heptose II        residue of the inner core bears in position O-6 or O-7 a        phosphoethanolamine (PEA) substituent and does not bear a PEA        substituent in position O-3,-   3) A vaccine composition according to the invention which comprises:    -   (i) an LOS obtained according to preparation process (i) or        an N. meningitidis LOS in particular constituted by a lipid A,        an inner core, an a chain of L6 type, in which the heptose II        residue of the inner core bears in position O-3 a        phosphoethanolamine (PEA) substituent and does not bear a PEA        substituent in positions O-6 and O-7; and    -   (ii) an N. meningitidis LOS in particular constituted by a lipid        A, an inner core, an α chain of L8 type, in which the heptose II        residue of the inner core bears in position O-3 a        phosphoethanolamine (PEA) substituent and does not bear a PEA        substituent in positions O-6 and O-7,-   4) A vaccine composition according to the invention which comprises:    -   (i) an LOS obtained according to preparation process (i) or        an N. meningitidis LOS in particular constituted by a lipid A,        an inner core, an a chain of L6 type, in which the heptose II        residue of the inner core bears in position O-3 and in position        O-6 or O-7 a phosphoethanolamine (PEA) sub stituent; and    -   (ii) an N. meningitidis LOS in particular constituted by a lipid        A, an inner core, an α chain of L8 type, in which the heptose II        residue of the inner core bears in position O-3 a        phosphoethanolamine (PEA) substituent and does not bear a PEA        substituent in positions O-6 and O-7,-   5) A vaccine composition according to the invention, which    comprises:    -   (i) an LOS obtained according to preparation process (iii) or        an N. meningitidis LOS in particular constituted by a lipid A,        an inner core, an a chain of L8 type, in which the heptose II        residue of the inner core bears in position O-3 and in position        O-6 or O-7 a phosphoethanolamine (PEA) sub stituent; and    -   (ii) an LOS obtained according to preparation process (ii) or        an N. meningitidis LOS in particular constituted by a lipid A,        an inner core, an a chain of L6 type, in which the heptose II        residue of the inner core bears in position O-3 a        phosphoethanolamine (PEA) substituent and does not bear a PEA        substituent in positions O-6 and O-7,-   6) A vaccine composition according to the invention, which    comprises:    -   (i) an LOS obtained according to preparation process (iii) or        an N. meningitidis LOS in particular constituted by a lipid A,        an inner core, an a chain of L8 type, in which the heptose II        residue of the inner core bears in position O-3 and in position        O-6 or O-7 a phosphoethanolamine (PEA) sub stituent; and    -   (ii) an LOS obtained according to preparation process (ii) or        an N. meningitidis LOS in particular constituted by a lipid A,        an inner core, an a chain of L6 type, in which the heptose II        residue of the inner core bears in position O-3 and in position        O-6 or O-7 a phosphoethanolamine (PEA) substituent.

A vaccine/pharmaceutical composition according to the invention isespecially useful for treating or preventing an infection caused by N.meningitidis, such as meningitis caused by N. meningitidis,meningococcemias and complications that may derive therefrom such aspurpura fulminans and septic shock; and also arthritis and pericarditiscaused by N. meningitidis.

It may be manufactured in a conventional manner. In particular, atherapeutically or prophylactically effective amount of the essentialconstituent of the vaccine, which is the LOS, is combined with apharmaceutically acceptable support or diluent. Advantageously, it mayalso comprise a pharmaceutically acceptable adjuvant.

For use in a composition according to the invention, the LOS(s) (are)advantageously formulated as liposomes.

Additionally, a composition according to the invention may comprise oneor more additional vaccine antigens of N. meningitidis; for example oneor more N. meningitidis polypeptides. In a particularly preferred form,a composition according to the invention may comprise (i) the subunit B(TbpB) of the human transferrin receptor, which is an outer membranelipoprotein of a certain number of non-enteric Gram-negative bacteriasuch as Neisseriae, e.g. N. meningitidis; or (ii) a lipidated N-terminalfragment thereof. In the latter case the lipidated TbpB or a lipidatedfragment thereof may act both as a vaccine antigen and as an LOSadjuvant.

In N. meningitidis, the strains are divided into two isotypes: isotypesI and II, which differ according to the length of the TbpB amino acidchain (EP 560 969 and EP 586 266). With regard to isotype I, thereference strain is strain B16B6. With regard to isotype II, thereference strain is strain M982.

According to one advantageous embodiment, a composition according to theinvention additionally comprises N. meningitidis isotype I or IIlipidated TbpB or lipidated TbpB of each of isotype I and II strains. Tothis end, the lipidated TbpB of N. meningitidis isotype I strain may bethat of strain B16B6; and the lipidated TbpB of N. meningitidis isotypeII strain may be that of strain M982.

Therefore, it is cited as a matter of example:

-   1) A vaccine composition of the invention, which comprises:    -   (i) a LOS obtained according to the preparation process (ii) or        a N. meningitidis LOS in particular in particular constituted by        a lipid A, an inner core, an α chain of L6 type, in which the        heptose II residue of the inner core bears in position O-3 a        phosphoethanolamine (PEA) substituent and does not bear a PEA        substituent in positions O-6 and O-7; and    -   (ii) the lipidated TbpB of a N. meningitidis strain of isotype        II or a lipidated N-terminal fragment thereof-   2) A vaccine composition of the invention, which comprises:    -   (i) a LOS obtained according to the preparation process (ii) or        a N. meningitidis LOS in particular in particular constituted by        a lipid A, an inner core, an α chain of L6 type, in which the        heptose II residue of the inner core bears in position O-3 a        phosphoethanolamine (PEA) substituent and does not bear a PEA        substituent in positions O-6 and O-7; and    -   (ii) the lipidated TbpB of a N. meningitidis strain of isotype        II or a lipidated N-terminal fragment thereof ; and    -   (iii) the lipidated TbpB of a N. meningitidis strain of isotype        I or a lipidated N-terminal fragment thereof-   3) A vaccine composition of the invention, which comprises:    -   (i) a LOS obtained according to the preparation process (ii) or        a N. meningitidis LOS in particular in particular constituted by        a lipid A, an inner core, an α chain of L6 type, in which the        heptose II residue of the inner core bears in position O-3 a        phosphoethanolamine (PEA) substituent and does not bear a PEA        substituent in positions O-6 and O-7; and    -   (ii) a N. meningitidis LOS in particular in particular        constituted by a lipid A, an inner core, an α chain of L8 type,        in which the heptose II residue of the inner core bears in        position O-3 a phosphoethanolamine (PEA) substituent and does        not bear a PEA substituent in positions O-6 and O-7; or A LOS        obtained according to the preparation process (iii); or a N.        meningitidis LOS in particular in particular constituted by a        lipid A, an inner core, an α chain of L8 type, in which the        heptose II residue of the inner core bears in position O-3 and        in position O-6 or O-7, a phosphoethanolamine (PEA) substituent;        and    -   (iii) the lipidated TbpB of a N. meningitidis strain of isotype        II or a lipidated N-terminal fragment thereof; and optionally    -   (iv) the lipidated TbpB of a N. meningitidis strain of isotype I        or a lipidated N-terminal fragment thereof.

When a vaccine composition of the invention comprises one or moreTbpB(s) this (these) latter can be formulated with the LOS in liposomesor be simply mixed with liposomes LOS (LOS formulated in liposomes).

The amounts of LOS per vaccine dose which are sufficient to achieve theabovementioned aims, and which are effective from an immunogenic,prophylactic or therapeutic point of view, depend on certain parametersthat include the individual treated (adult, adolescent, child orinfant), the route of administration and the administration frequency.

The amount of LOS per dose which is sufficient to achieve theabovementioned aims is in particular between 5 and 500 μg,advantageously between 10 and 200 μg, preferably between 20 and 100 μg,entirely preferably between 20 and 80 μg or between 20 and 60 μg, limitsincluded.

The term “dose” employed above should be understood to denote a volumeof vaccine administered to an individual in one go—i.e. at a time T.Conventional doses are of the order of a milliliter, for example 0.5, 1or 1.5 ml; the definitive choice depending on certain parameters, and inparticular on the age and the status of the recipient. An individual canreceive a dose divided up into injections at several injection sites onthe same day. The dose may be a single dose or, if necessary, theindividual may also receive several doses a certain time apart—it beingpossible for this time apart to be determined by those skilled in theart.

It may be administered by any conventional route in use in the priorart, e.g. in the vaccines field, in particular enterally orparenterally. The administration may be carried out as a single dose oras repeated doses a certain time apart. The route of administrationvaries as a function of various parameters, for example of theindividual treated (condition, age, etc.).

Finally, the invention also relates to:

-   -   a method for inducing in a mammal, for example a human, an        immune response against N. meningitidis, according to which an        immunogenically effective amount of a composition according to        the invention is administered to the mammal so as to induce an        immune response, in particular a protective immune response        against N. meningitidis; and    -   a method for prevention and/or treatment of an infection caused        by N. meningitidis, according to which a prophylactically or        therapeutically effective amount of a composition according to        the invention is administered to an individual in need of such a        treatment.

Experimental Data

A Experimental Data Relating to the Strains Derived from N. meningitidisC708

1. Materials & Methods 1.1 Transformation of Strain C708

Strain C708 is cultured in BHI (Brain Heart Infusion) agar medium at 37°C. under an atmosphere containing 10% CO₂. The bacterial lawn isharvested in BHI liquid medium complemented with 5 mM MgCl₂ to obtain abacterial suspension at 10⁹ cfu/ml (cfu: colony-forming unit).

To 900 μl of the BHI liquid medium +5 mM MgCl₂ are added 10 μg of DNAnecessary for the transformation (linearized plasmid); followed by 100μl of the bacterial suspension (10⁸ microorganisms). The transformationmedium is incubated for 30 minutes at 37° C., 10% CO₂.

500 μl of this preparation (i.e. about 5.10⁷ cfu) serve to inoculate 4.5ml of BHI +5 mM MgCl₂. The bacteria are left to regenerate for 2 hoursat 37° C., 10% CO₂. Next, starting with this suspension, dilutions ofBHI+5 mM MgCl₂ are made. 300 μl of a dilution containing about 30 000cfu are plated out on a 140 mm agar BHI dish. The dishes are placed at37° C., 10% CO₂ for at least 20 hours.

1.2. Blotting of the Transformant Colonies

The colonies are transferred onto 137 mm Hybond-XL membrane (GEHealthcare; #RPN 137S). The microorganisms deposited on the membrane arelysed in denaturing buffer (0.5 M NaOH, 1.5 M NaCl). The membranes arewashed with neutralizing buffer (0.5 M Tris, 1.5 M NaCl, pH 7.5);transferred into SSC 2× medium; and then dried. The DNA is bound byincubation for 2 hours at 80° C.

1.3. Detection of the Transformants by Hybridization with a ProbeLabeled with ³³P dCTP

Labeling of the probe is obtained by PCR amplification using theReady-to-Go PCR Beads kit (GE Healthcare); the labeled probe is thenpurified on a ProbeQuant G50 Microcolumn column (GE Healthcare).

The membranes to be hybridized are placed in threes in 50 ml of RapidHyb buffer (GE Healthcare) for 15 minutes at 65° C., with slow stirring,for prehybridization. The probe labeled and denatured beforehand for 2minutes at 95° C. is added to the membranes. The final probeconcentration is 5 ng/ml. Hybridization is allowed to continue for 2hours at 65° C., with slow stirring.

The membranes are then subjected to successive washes by working asfollows:

-   -   in low stringency buffer (2×SSC, 0.1% SDS (weight/vol)) 15        minutes at ambient temperature with slow stirring;    -   in medium stringency buffer (1×SSC, 0.1% SDS (weight/vol)) 20        minutes at 65° C. with slow stirring; and    -   low stringency buffer (0.1×SSC, 0.1% SDS (weight/vol)) 45        minutes at 65° C. with slow stirring.

Once dried, the membranes are revealed by autoradiography (Biomax MRfilm).

1.4. Detection of the Transformants by Hybridization with anOligonucleotide Labeled with [γ³²P] ATP

Labeling of the 5′ end of the oligonucleotide is performed in thefollowing reaction medium (the amounts indicated are those correspondingto the hybridization of an amount of oligonucleotide necessary for thehybridization of 3 membranes in a dish):

free 5′-OH oligonucleotide 3 μl max i.e. 10 pmol 10 X phosphorylationbuffer 1 μl i.e. 1X [γ-³²P] ATP 10 mCi/ml 5 μl i.e. 50 μCi T4 kinase (10U/μl) 1 μl i.e. 10 U H₂O qs 10 μl

The reaction medium is incubated at 37° C. for 30 minutes. Next, the T4kinase is inactivated by heating for 10 minutes at 70° C.

The membranes to be hybridized are placed in threes in 60 ml of RapidHyb buffer (GE Healthcare) for 15 minutes at 48° C., with slow stirring,for prehybridization. The prehybridization buffer is removed andreplaced with 50 ml of hybridization buffer as follows: 5×SSC,5×Denhardt's solution, 0.5% SDS (weight/vol.) and 100 μg/ml of salmonsperm DNA at 10 mg/ml sonicated and denatured for 5 minutes at 100° C.

The labeled oligonucleotide (10 μl) is added to the membranes. Thehybridization is allowed to continue overnight at a temperature 5° C.below the Tm of the oligonucleotide, with gentle stirring.

The membranes are then subjected to successive washes by working in theorder as follows:

-   -   in low stringency buffer (2×SSC, 0.1% SDS (weight/vol) 5 minutes        at the Tm of the oligonucleotide −5° C., with slow stirring;    -   in medium stringency buffer (1×SSC, 0.1% SDS (weight/vol) 15        minutes at the Tm of the oligonucleotide −5° C., with slow        stirring; and    -   in low stringency buffer (0.1×SSC, 0.1% SDS (weight/vol) 10        minutes at the Tm of the oligonucleotide −5° C., with slow        stirring.

Once dried, the membranes are revealed by autoradiography (Biomax MRfilm).

2. Construction of a Strain of N. meningitidis Expressing an LOS havingan a Chain is that of an LOS of Immunotype L6 and Comprising in Each ofthe Positions O3 and O6 of the Heptose II (hep II) Residue of the InnerCore a Phosphoethanolamine (PEA) Substituent

The starting strain used is N. meningitidis strain C708 of serogroup Aand immunotype L6 having, inter alia, the following characteristics:

-   -   an active lgtA gene (gene switched “ON”);    -   an lgtB gene—(non-functional gene);    -   an inactive lgtG gene (switched “OFF”);    -   a truncated lpt3 gene;    -   an active lpt6 gene; and    -   an active lot3 gene.

Strain C708 was filed on Mar. 11, 2008 at the Collection Nationale deCulture de Microorganisme, 25 rue du Dr Roux 75015 Paris, according tothe terms of the treaty of Budapest. This strain bears the order numberCNCM 1-3942.

Strain C708 comprises a truncated lpt3 gene. To modify it such that theLOS bears a PEA substituent in position O3, it is chosen to replace byhomologous recombination the truncated lpt3 gene with the complete(full-length) lpt3 gene of the strain of N. meningitidis FAM18 serogroupC (strain made available worldwide to research laboratories). The strainresulting therefrom will be referred to for greater convenience as C708lpt3 FL.

2.1. PCR (Polymerase Chain Reaction) Amplification of the Full-Length(FL) lpt3 Gene of N. meningitidis strain FAM18

100 ng of genomic DNA of the strain FAM18 were used for amplificationwith Platinum® Taq DNA polymerase High Fidelity (Invitrogen,#11304-011).

The pair of primers is as follows (pair No. 1):

(SEQ ID NO: 8) CG GAATTC GCC GTC TCA A ATG AAA AAA TCC CTT TTC GTT CTC(Tm = 55.9° C.); and (SEQ ID NO: 9)AA CTGCAG TCA TTG CGG ATA AAC ATA TTC CG (Tm = 57.1° C.);(the EcoRI and PstI sites are respectively underlined).

The following mixture was used for amplification:

Components Volume Final concentration 10X High Fidelity PCR buffer 5 μl1X 10 mM dNTP mixture 1 μl 0.2 mM of each 50 mM MgSO₄ 2 μl 2 mM Mixtureof primers (10 μM each) 1 μl 0.2 μM of each Genomic DNA x μl 100 ngPlatinum ® Taq High Fidelity 0.2 μl 1.0 unit Nuclease-free water qs 50μl final Does not apply

The thermocycler program is as follows:

Initial denaturing: 94° C. for 30 seconds 30 cycles of: denaturing: 94°C. for 30 seconds hybridization: 55° C. for 30 seconds extension: 68° C.for 1 minute/kb of PCR product.

After the reaction, 1/10 of the PCR product was deposited on agarose gelfor verification.

2.2. Construction of a Transformation Vector

The PCR product, on the one hand, and the plasmid pUC19, on the otherhand, were subjected to double digestion with EcoRI and PstI for 2 hoursat 37° C. 10 units of each enzyme per μg of DNA in REact2 buffer(Invitrogen) were used.

The PCR fragment was then inserted into the linearized pUC19 vector. Theligations were performed in a final volume of 20 μl with 50 ng ofvector, 0.5 U of T4 DNA ligase (Invitrogen) and 1 μl of 10 mM ATP(Invitrogen) for 16 hours at 16° C. The ligase was then inactivated byheating for 10 minutes at 65° C.

The vector thus obtained was transferred via the electroporationtechnique into a strain of E. coli XLI blue MRF resistant to kanamycinand made electrocompetent. The parameters adopted for theelectroporation are as follows: capacitance: 500 μFD; resistance: 200ohms; voltage: 1700 volts.

Selection of the transformed clones was performed by plating out on 100μg/ml ampicillin LB dishes. Authentification of 10 of the 50 positiveclones was performed by PCR amplification. 100% of the clones had theexpected profile. Finally, the lpt3 gene in a plasmid of one of theseclones (plasmid pM1222) was verified by sequencing.

2.3. Transformation of Strain C708 and Detection of the HomologousRecombination Event 2.3.1. Transformation

40 μg of pM1222 were digested with 400 U of EcoRI and 10 μg were used totransform the strain C708 according to the method described in sectionA.1.1.

After transformation, the bacteria were plated out onto 17 140 mm Petridishes at a theoretical concentration of 30 000 cfu per dish; i.e. 510000 cfu (colony-forming units) and were then placed overnight at 37° C.The dishes were then placed at +4° C. for 30 minutes.

After 24 hours at 37° C., counting of the control dishes made itpossible to estimate the number of cfu as 27 000 per dish for themutant.

2.3.2. Clone Selection

The recombination event, i.e. the replacement of the lpt3 TR (truncated)gene with the lpt3 FL (full-length) gene, was detected aftertransferring the clones onto hybridization membrane and hybridizing witha probe labeled with ³³P dCTP according to the methods described insections A.1.2. and A.1.3.

Selection of the positive clones was made by hybridization of the DNAbound to the membranes with a DNA probe labeled with ³³P dCTPcorresponding to the truncated part of the lpt3 gene, which is thuspresent only in the recombinant clones.

Preparation of the Probe

In order to obtain the 270-bp lpt3 probe, 10 ng of the plasmid pUClpt3were used for PCR amplification with Platinum® Taq DNA polymerase HighFidelity (Invitrogen, #11304-011).

The pair of primers is as follows (pair No. 2):

(SEQ ID NO: 10) CGC CGA ATA CTT TAT CTT GAG GC (Tm = 60.6° C.); and(SEQ ID NO: 11) CTC GCC AAA GAG CAG GGC (Tm = 60.5° C.).

For amplification, the following mixture was used:

Components Volume Final concentration 10X High Fidelity PCR buffer 5 μl1X 10 mM dNTP mixture 1 μl 0.2 mM of each 50 mM MgSO₄ 2 μl 2 mM Mixtureof primers (10 μM each) 1 μl 0.2 μM of each Plasmid pUC x μl 100 ngPlatinum ® Taq High Fidelity 0.2 μl 1.0 unit Nuclease-free water qs 50μl final Does not apply

The thermocycler program is as follows:

Initial denaturing: 94° C. for 30 seconds 30 cycles of: Denaturing: 94°C. for 30 seconds Hybridization: 55° C. for 30 seconds Extension: 68° C.for 45 seconds.

After the reaction, 1/10 of the PCR products was deposited on agarosegel to ensure the specificity of the amplicon, and the PCR fragment wasthen purified using the QlAquick PCR Purification Kit (Qiagen, #28104).

Hybridization and Revelation

The steps of labeling of the probe, hybridization, washing andrevelation were performed as described in section A.1.3.

About 460 000 cfu were tested. After exposure with the BioMax MR films,the autoradiographs revealed 5 positive spots (C708 containing an lpt3FL gene) each on a different membrane.

Screening and Authentification of the Positive Clones

After locating on the Petri dish, part of the zone taken up around thepositive clones was stored in freezing medium (M199 medium, 20% fetalcalf serum, 10% glycerol) and the other part was used for the PCRauthentification.

To do this, each of the samples collected was first taken up in 80 μl ofBHI broth, so as to plate out 30 μl of this suspension, as a mini-lawnon a BHI dish.

The remaining volume was centrifuged for 5 minutes at 6000 rpm and thepellet was then taken up in 50 μl of nuclease-free water. Themicroorganisms were lysed for 5 minutes at 95° C. and the supernatant,which serves as the matrix for the PCR reaction, was collected aftercentrifugation.

For each collected sample corresponding to a positive spot and for thecontrols, PCR amplification with the pair of primers that served for theamplification of the 270 bp C708 lpt3 probe (pair No. 2) was performedwith the Expand Long Template PCR kit (Roche) as described below.

Components Volume Final concentration 10X ELT PCR buffer 5 μl 1X dNTPmixture (10 mM of each) 2 μl 0.4 mM of each mixture of primers (10 μM ofeach) 1.5 μl 0.3 μM of each DNA matrix 40 μl Does not apply PolymeraseELT 0.75 μl 3.75 units Nuclease-free water qs 50 μl Does not apply

The thermocycler program is as follows:

Initial denaturing: 94° C. for 2 min 10 cycles of: denaturing: 94° C.for 10 seconds hybridization: 54° C. for 30 seconds extension: 68° C.for 45 seconds 20 cycles of: denaturing: 94° C. for 15 secondshybridization: 54° C. for 30 seconds extension: 68° C. for 45 seconds +20 sec/cycle Final elongation: 68° C. for 7 min

After the reaction, 1/10 of the PCR products was deposited on agarosegel for verification. Four of the 5 clones had the expected profile. Thefrequency of production of a true positive clone was 1/115000 cfutested.

The following step consisted in isolating a pure clone. To do this, oneof the heterogeneous positive clones was plated out as isolated cfus andseveral of these cfus (40) were analyzed by PCR, with the pairs ofprimers 1 or 2.

Each cfu was resuspended in 100 μl of nuclease-free water, 30 μl weredeposited on a BHI dish and the remaining 70 μl were lysed for 5 minutesat 95° C., and the supernatant, which serves as the matrix for the PCRreaction, was collected after centrifugation.

The PCRs were performed with Platinum® Taq High Fidelity (Invitrogen) asalready described for the amplification of the lpt3 probe. Thehybridization temperature was 54° C.

After the reaction, 1/10 of the PCR products was deposited on agarosegel for verification. Five of the 40 clones proved to be pure clones.

The mini-lawn of pure clones was taken up in freezing medium, dividedinto 100 μl aliquots and stored at −70° C. The purity and the identityof this freezing material were validated.

3. Construction of a Strain of N. meningitidis Expressing an LOS Havingan a Chain is that of an LOS of Immunotype L6 and Comprising Only inPosition O3 of the Heptose II (hep II) Residue of the Inner Core aPhosphoethanolamine (PEA) Substituent

The starting strain used is N. meningitidis strain C708 lpt3 FL obtainedas described previously. The objective is to inactivate the lpt6 gene ofthis strain by deletion of a central part of the gene.

3.1. PCR (Polymerase Chain Reaction) Amplification of the Full-Lengthlpt6 Gene of N. meningitidis strain Z2491 of Serogroup A (gene NMA 0408)

100 ng of genomic DNA of strain Z2491 (strain made available worldwiseto research laboratories) were used for amplification with Platinum® TaqDNA polymerase High Fidelity (Invitrogen, #11304-011).

The pair of primers is as follows (pair No. 3):

(SEQ ID NO: 12) CG GAATTC GCC GTC TCA A GGT TGC CTA TGT TTT CCTGTT TTT G (Tm = 59.7° C.); and (SEQ ID NO: 13)AA CTGCAG CTA ACG GGC AAT TTT CAA AAC GTC (Tm = 59.3° C.);(the EcoRI and PstI sites are respectively underlined).

For amplification the following mixture was used:

Components Volume Final concentration 10X High Fidelity PCR buffer 5 μl1X 10 mM dNTP mixture 1 μl 0.2 mM of each 50 mM MgSO₄ 2 μl 2 mM Mixtureof primers (10 μM each) 1 μl 0.2 μM of each Genomic DNA x μl 100 ngPlatinum ® Taq High Fidelity 0.2 μl 1.0 unit Nuclease-free water qs 50μl final Does not apply

The thermocycler program is as follows:

Initial denaturing: 94° C. for 30 seconds 30 cycles of: denaturing: 94°C. for 30 seconds hybridization: 55° C. for 30 seconds extension: 68° C.for 1 minute/kb of PCR product.

After the reaction, 1/10 of the PCR product was deposited on agarose gelfor verification.

3.2. Construction of Vector pM1223 (pUC19 lpt6 FL)

The PCR product, on the one hand, and plasmid pUC19, on the other hand,were subjected to double digestion with EcoRI and PstI for 2 hours at37° C. 10 units of each enzyme per μg of DNA were used in the bufferREact2 (Invitrogen).

The PCR fragment was then inserted into the linearized pUC19 vector.Ligations were performed on a final volume of 20 μl with 50 ng ofvector, 0.5 U of T4 DNA ligase (Invitrogen) and 1 μl of 10 mM ATP(Invitrogen) for 16 hours at 16° C. The ligase was then inactivated byheating for 10 minutes at 65° C.

The vector thus obtained was transferred via the electroporationtechnique into a strain of E. coli XL1 blue MRF resistant to kanamycinand made electrocompetent. The parameters adopted for theelectroporation are as follows: capacitance: 500 μFD; resistance: 200ohms; voltage: 1700 volts.

Selection of the transformed clones was performed by plating out onto100 μl g/ml ampicillin LB dishes. Authentification of the positiveclones (presence of an lpt6 FL gene) was performed by NdeI enzymaticdigestion after extraction of the DNA by miniprep. Out of 20 clonesanalyzed, 6 had the expected profile. The recombinant plasmid of theselected clone was named pM1223.

3.3. Deletion of the Central Part of the lpt6 Gene Originating fromStrain Z2491 Construction of a Transformation Vector

With the Expand Long Template PCR kit (Roche), a reverse PCR wasperformed using the plasmid pM1223 with the aid of the following pair ofprimers (pair No. 4):

(SEQ ID NO: 14) CG GGATCC CAT CGA CAC GAA CGC CGC (Tm = 60.5° C.); and(SEQ ID NO: 15) CG GGATCC CCG CGC TTA ACG ACT ACA TC (Tm = 59.4° C.);(the BamHI sites are underlined).

This makes it possible to amplify again the plasmid while deleting thepart that it is desired to remove (808 bp).

The following mixture was used for amplification:

Components Volume Final concentration 10X ELT PCR buffer 5 μl 1X dNTPmixture (10 mM of each) 2 μl 0.4 mM of each Mixture of primers (10 μM ofeach) 1.5 μl 0.3 μM of each DNA matrix (pM1223) 40 μl Does not applyPolymerase ELT 0.75 μl 3.75 units Nuclease-free water qs 50 μl Does notapply

The thermocycler program is as follows:

Initial denaturing: 94° C. for 2 minutes 10 cycles of: denaturing: 94°C. for 10 seconds hybridization: 54° C. for 30 seconds extension: 68° C.for 3 minutes 20 cycles of: denaturing: 94° C. for 15 secondshybridization: 54° C. for 30 seconds extension: 68° C. for 3 minutes +20 sec/cycle Final elongation: 68° C. for 7 minutes

After the reaction, 1/10 of the PCR products was deposited on agarosegel.

After purification on a QiaQuick column, the PCR product was digestedwith BamHI at a rate of 10 U of enzyme per μg of DNA. Once digested, itwas purified by electro-elution and then extracted withphenol-chloroform.

Self-ligation of the vector was performed in a final volume of 20 μlwith 0.5 U of T4 DNA ligase (Invitrogen) and 1 μl of 10 mM ATP(Invitrogen) for 16 hours at 16° C. The ligase was then inactivated byheating for 10 minutes at 65° C.

The final step consisted in transferring the vector thus ligated into E.coli as described for pM1222. Authentification of the positive cloneswas performed by NdeI-PstI enzymatic digestion after extraction of theDNA by miniprep. Out of the 4 clones analyzed, 100% had the expectedprofile.

The recombinant plasmid of the selected positive clone was named pM1224,and this clone was stored in glycerol at −70° C. The presence in theplasmid pM1224 of an lgt6 gene with its central part deleted wasconfirmed by sequencing.

3.4. Transformation of Strain C708 lpt3 FL and Detection of theHomologous Recombination Event

Transformation

10 μg of plasmid pM1224 were linearized with EcoRI at a rate of 10 unitsof enzyme per of plasmid to be digested in the appropriate buffer for 2hours at 37° C.

The transformation of strain C708 with plasmid pM1224 was performedaccording to the technique described in section A.1.1.

After transformation, the bacteria were plated out on 16 140 mm Petridishes at a theoretical concentration of 50 000 cfu per dish; i.e. 800000 cfu. The dishes were placed overnight at 37° C. and then placed for30 minutes at +4° C.

Clone Selection: Preparation of the Probe, Hybridization and Revelation

The recombination event was detected after colony blotting andhybridization with an oligonucleotide labeled with γ³²P dATP.

The recombination event, i.e. the replacement of the lpt6 FL gene withthe lpt6 TR gene, was detected after transferring the clones ontomembrane and hybridization with a labeled probe according to the methodsdescribed in sections A.1.2. and A.1.4.

The clones transferred onto membranes were subjected to lysis andwashing steps. The DNA is bound to the membranes by placing them for 2hours at 80° C.

Selection of the positive clones was performed by hybridization of theDNA bound to Hybond N+ membranes with a radioactive oligonucleotidewhose sequence overlaps the two recombigenic ends. This is the followingoligonucleotide: GTC GAT GGG ATC CCC GCG CTT AAC G (SEQ ID NO: 16)(Tm=69.5° C.).

About 840 000 cfu were tested. After exposure with BioMax MR films, theautoradiographs revealed 16 positive spots (C708 containing an lpt6 TRgene) divided among 9 different membranes.

Screening and Authentification of the Positive Clones

For each of the 16 positive spots, after detection on the Petri dish,part of the zone collected around the positive clones was stored infreezing medium (M199, 20% FCS, 10% glycerol) and the other part wasused for the PCR authentification.

To do this, the collected samples were first taken up in 80 μl of BHIbroth, so as to plate out, as a mini-lawn on a BHI dish, 30 μl of eachsuspension.

The remaining volume was centrifuged for 5 minutes at 6000 rpm, and thepellet was then taken up in 50 μl of nuclease-free water. Themicroorganisms were lysed for 5 minutes at 95° C. and the supernatant,which serves as the PCR reaction matrix, was collected aftercentrifugation.

For each collected sample corresponding to a positive spot, a PCRamplification PCR was performed with the Platinum® Taq High Fidelity kit(Invitrogen) and the following pair of primers (pair No. 5)

(SEQ ID NO: 17) CCG ACT GGC GGA ATT GGG (TM = 60.5° C.); and(SEQ ID NO: 18) CCC ATT TCT TCC TGA CGG AC (Tm = 59.4° C.).

The following mixture was used for amplification:

Components Volume Final concentration 10X High Fidelity PCR buffer 5 μl1X 10 mM dNTP mixture 1 μl 0.2 mM of each 50 mM MgSO₄ 2 μl 2 mM Mixtureof primers (10 μM each) 1 μl 0.2 μM of each DNA matrix x μl 100 ngPlatinum ® Taq High Fidelity 0.2 μl 1.0 unit Nuclease-free water qs 50μl final Does not apply

The thermocycler program is as follows:

Initial denaturing: 94° C. for 1 minute 30 cycles of: denaturing: 94° C.for 30 seconds hybridization: 55° C. for 30 seconds extension: 68° C.for 50 seconds.

After the reaction, 1/10 of the PCR product was deposited on agarosegel, for verification. Two candidates out of 16 proved to be truepositives: i.e. a frequency of production of one true positive clone per425 000 cfu tested.

The following step consisted in isolating a pure clone. To do this, oneof the 2 hetero-geneous positive clones was plated out as isolated cfusand several of these cfus (24) were analyzed by PCR, with the pair ofprimers No. 5.

Each cfu was resuspended in 50 μl of nuclease-free water, 20 μl weredeposited on a BHI dish and the remaining 30 μl were lysed for 5 minutesat 95° C. and the supernatant, which serves as PCR reaction matrix, wascollected after centrifugation.

The PCRs were performed with the Platinum® Taq High Fidelity kit(Invitrogen) as already described for the screening of the collectedsamples of the positive spots.

After the reaction, ⅕ of the PCR products was deposited on agarose gelfor verification. Only one clone out of 24 proved to be a true positive.

The mini-lawn of the pure positive clone was taken up in freezingmedium, divided into 100 μl aliquots and stored at −70° C. The purityand identity of this frozen material were confirmed.

4. Construction of a N Meningitidis Strain Expressing an LOS Having an αChain which is That of an LOS of Immunotype L8 And Comprising a SinglePhosphoethanolamine (Pea) Substituent in Position O3 Of The Heptose II(Hep II) Residue of The Inner Core

N. meningitidis strain C708 lpt3 FL obtained as described previously insection A.3. is used as starting strain. The goal is to inactivate thelgtA gene of this strain by deletion of a central part of the gene.

4.1. PCR (Polymerase Chain Reaction) Amplification of the Full-LengthlgtA Gene of N. meningitidis Strain MC58 of Serogroup B (gene NMB 1929)

100 ng of genomic DNA of strain MC58 (strain made available worldwise toresearch laboratories) were used for amplification with Platinum® TaqDNA polymerase High Fidelity (Invitrogen, #11304-011).

The pair of primers is as follows:

(SEQ ID NO: 19) CG GAATTC GCC GTC TCA A ATG CCG TCT GAA GCC TTC AG (Tm =59.4° C.); and (SEQ ID NO: 20) AA CTGCAG AAC GGT TTT TCA GCA ATC GGT(Tm = 60.6° C.);(the EcoRI and PstI sites are respectively underlined).

For amplification the following mixture was used:

Components Volume Final concentration 10X High Fidelity PCR buffer 5 μl1X 10 mM dNTP mixture 1 μl 0.2 mM of each 50 mM MgSO₄ 2 μl 2 mM Mixtureof primers (10 μM each) 1 μl 0.2 μM of each Genomic DNA x μl 100 ngPlatinum ® Taq High Fidelity 0.2 μl 1.0 unit Nuclease-free water qs 50μl final Does not apply

The thermocycler program is as follows:

Initial denaturing: 94° C. for 30 seconds 30 cycles of: denaturing: 94°C. for 30 seconds hybridization: 55° C. for 30 seconds extension: 68° C.for 1 minute/kb of PCR product.

After the reaction, 1/10 of the PCR product was deposited on agarose gelfor verification.

4.2. Construction of Vector pUC19 lgtA FL)

The PCR product obtained in A.4.1., on the one hand, and plasmid pUC19,on the other hand, were submitted to a double digestion with EcoRI andPstI for 2 hours at 37° C. 10 units of each enzyme per μg of DNA wereused in the buffer REact2 (Invitrogen).

The PCR fragment was then inserted into the linearized pUC19 vector.Ligations were performed under a final volume of 20 μl with 50 ng ofvector, 0.5 U of T4 DNA ligase (Invitrogen) and 1 μl of 10 mM ATP(Invitrogen) for 16 hours at 16° C. The ligase was then inactivated byheating for 10 minutes at 65° C.

The vector thus obtained was transferred by the electroporationtechnique into E. coli strain XL1 blue MRF kanamycin-resistant and madeelectrocompetent. The parameters adopted for the electroporation are asfollows: capacitance: 500 μFD; resistance: 200 ohms; voltage: 1700volts.

Selection of the transformed clones was performed by plating out onto100 μg/ml ampicilline LB dishes. Authentification of the positive clones(presence of an lgtA FL gene) was performed by NdeI enzymatic digestionafter extraction of the DNA by miniprep. ⅕ of the analyzed clones hadthe expected profile.

4.3. PCR Amplification of the Erythromycine (erm) Gene

PCR amplification of the erythromycine cassette (erm) was achieved usingplasmid pMCG10 as template with primers allowing the introduction ofrestriction sites BamHI and XbaI.

The primer pair is as follows:

(SEQ ID NO: 21) CG GGATCC GGA AGG CCC GAG CGC AGA AGT (Tm: 65.7° C.);and (SEQ ID NO: 22) GC TCT AGA CAA CTT ACT TCT GAC AAC GAT CGG (Tm: 61°C.)

For amplification the following mixture was used:

Components Volume Final concentration 10X Pfu turbo buffer 5 μl 1X 10 mMdNTP mixture 0.4 μl 0.2 mM of each 50 mM MgSO₄ 2 μl 2 mM Mixture ofprimers (10 μM each) 1 μl 0.2 μM of each pMGC10 x μl 10 ng Pfu turbo(Stratagene) 0.2 μl 2.5 units Nuclease-free water qs 50 μl final Doesnot apply

The thermocycler program is as follows:

Initial denaturing: 95° C. for 30 seconds 30 cycles of: denaturing: 95°C. for 30 seconds hybridization: 55° C. for 30 seconds extension: 72° C.for 10 min.4.4. Construction of Plasmid pUC19 lgtA TR (Deletion of the Central Partof the lgtA Gene by Reverse PCR)

With the Expand Long Template PCR kit (Roche), a reverse PCR wasachieved using plasmid pUC19 with the double objective of removing thecentral part of lgtA and of creating two restriction sites (BamHI andXbaI). The following pair of primers was used:

(SEQ ID NO: 23) CG GGATCC GCC AAT TCA TCC AGC CCG ATG (Tm = 61.8° C.);and (SEQ ID NO: 24) CG TCTAGA CCC GGT TCG ACA GCC TTG (Tm = 60.5° C.);(the BamHI and XbaI sites are underlined).

This makes it possible to amplify AGAIN the plasmid while deleting thepart that it is desired to remove.

The following mixture was used for amplification:

Components Volume Final concentration 10X ELT PCR buffer 5 μl 1X dNTPmixture (10 mM of each) 2 μl 0.4 mM of each Mixture of primers (10 μM ofeach) 1.5 μl 0.3 μM of each 10 ng of pUC19 lgtA FL Does not applyPolymerase ELT 0.75 μl 3.75 units Nuclease-free water qs 50 μl Does notapply

The thermocycler program is as follows:

Initial denaturing: 94° C. for 2 minutes 10 cycles of: denaturing: 94°C. for 10 seconds hybridization: 55° C. for 30 seconds extension: 68° C.for 1 minute per kbs 20 cycles of: denaturing: 94° C. for 15 secondshybridization: 55° C. for 30 seconds extension: 68° C. for 1 minute perkbs + 20 sec/cycle Final elongation: 68° C. for 7 minutes

After the reaction, 1/10 of the PCR products was deposited on agarosegel for verifying the size (3.2 kbs). The PCR product was purified on aQiaQuick column.

The final step has consisted in transferring the plasmid into E. colistrain XL1 blue MRF kanamycin-resistant and made electrocompetent asdescribed above e.g. for pM1222. Authentification of the positive clones(lgtA with a deleted central region) was performed by enzymaticdigestion after extraction of the DNA by miniprep.

4.5. Construction of Plasmid pUC19 lgtA::erm

The PCR product erm, on the one hand, and the plasmid pUC19 lgtA TRobtained by reverse PCR, on the other hand, were submitted to a doubledigestion with BamHI and XbaI under the following conditions:

2 μg of DNA were mixed with 20 units of XbaI in 60 μL of buffer 2(InVitrogen) for 2 hours 37° C. Then XbaI was inactivated by heating. 7μL of NaCl 1 M, 20 BamHI units and 1 μL of buffer 2 were added. Thereaction was performed for 2 hours at 37° C.

The digestion products were then deposited on a 0.8% agarose gel andafter migration, bands were cut out for further electroelution (plasmidband migrates at 3.2. kbs).

Upon purification, the linearized plasmid and the digested PCR producterm were ligated together under ligation conditions as describedpreviously. The ligation product has been used to transform aspreviously described, E. coli strain XL1 blue MRF kanamycin-resistantand made electro-competent. The recombinant clones were analyzed byenzymatic digestion. 4/11 of the analyzed clones had the expecteddigestion profile.

4.6. Transformation of Strain C708 lpt3 FL lpt6 TR and Detection of theHomologous Recombination Event 10 μg of plasmid pUC19 lgtA::erm werelinearized with EcoRI at a rate of 10 units of enzyme per μg of plasmidto be digested in the appropriate buffer for 2 hours at 37° C.

The transformation of strain C708 was performed according to thetechnique described in section A.1.1.

After transformation, 1.24 10⁸ bacteria were plated out on BHI+2 μg/mLerythromycine and incubated overnight at 37° C. The transformation ratewas 1/2.5 10⁶.

B. Experimental Data Relating to the Vaccine Compositions

1. Preparation of Lipidated rTbpB M982 and B16B6

In the interest of simplifying the language and reading, the term“rTbpB” or “TbpB” will subsequently be simply indicated.

1.1. Production

Strains Expressing rTbpB M982 and B16B6

The expression strains are E. coli BL21 strains respectively containingplasmids TG9219 and TG9216. These plasmids contain in particular akanamycin-selectable marker and the polynucleotide encoding the rTbpBfrom N. meningitidis strain M982 (pTG9219) or B16B6 (pTG9216) (thesequences of which are as described in patent EP 586 266) fused to theE. coli R1pB (real lipoprotein B) signal sequence and placed under thecontrol of the arabinose promoter (araB).

Culture

Three frozen samples of E. coli BL21/pTG9219 or E. coli BL21/pTG9216strain (each 1 ml) are used to inoculate 3 liters of LB (Luria Broth)medium divided up in Erlenmeyer flasks. The incubation is continued for15 to 18 h at 37° C.

This preculture is used to inoculate a fermenter containing TGM16 medium(9 g/L yeast extract, 0.795 g/L K₂SO₄, 3.15 g/L K₂HPO₄, 0.75 g/L NaCl,0.005 g/L CaCl₂2H₂O, 0.021 g/L FeCl₃.6H₂O, 0.69 g/L MgSO₄.7H₂O, 37.5 g/Lsalt-free casein acid hydrolysate) supplemented with 20 g/L glycerol, ina proportion of 10% (vol./vol).

The culturing is continued at 37° C. with shaking, at a pressure of 100mbar and with an air feed of 1 L/min/L of culture, while readjusting,over time, the glycerol concentration to 20 g/L (e.g. at OD₆₀₀ of 15±2).When the OD₆₀₀ is between 21 and 27, the rTbpB expression is induced byadding arabinose so as to obtain a final concentration of 10 g/L. Afterone hour of induction, the culture is stopped by cooling to around 10°C.

The bacterial pellets are recovered by centrifugation and stored in thecold.

1.2. Purification

Extraction of Membranes Containing the rTbpB

LOS Extraction

A bacterial pellet equivalent to one liter of culture (approximately 72g of microorganisms, wet weight) is thawed at a temperature of 20° C.+/−5° C. The thawed (or partially thawed) microorganisms are resuspendedwith 800 ml of a solution, at ambient temperature, of 50 mM Tris HCl, 5mM EDTA, pH 8.0. 9 protease inhibitor tablets (7 Complete Mini, EDTAfree tablets; ROCHE ref 11836170001+two Complete, EDTA free tablets;ROCHE ref. 11836170001) are immediately added. Since some of themicroorganisms lyze spontaneously, 4 μl of benzonase (1 IU of DNAseactivity/ml final concentration; Merck ref. K32475095) are also added.The incubation is continued at +4° C. for 45 minutes with magneticstirring after homogenization with a Turrax (15 sec.).

4 ml of 1 M MgCl₂ are then added so as to be at a final concentration of5 mM. The magnetic stirring is continued for 10 minutes. Centrifugationat 15 000 g for 45 minutes makes it possible to harvest the pellet(pellet P1; versus supernatant S1) containing the rTbpB protein.

A second extraction is carried out: homogenization with a Turrax in 800ml of the 50 mM Tris HCl buffer containing 5 mM EDTA, pH 8.0, andstirring for 30 min. MgCl₂ (8 ml of a molar solution) is added. Theincubation is continued for 10 minutes. The suspension is centrifuged at15 000 g for 1 hour 30.

Bacterial Lysis

The pellet is resuspended with 1400 ml of 50 mM Tris HCl supplementedwith 4 protease inhibitor tablets with 8 μl of benzonase. The solutionis homogenized with a Turrax for 15 seconds. The lysis is carried out at+4° C. for 30 minutes through the addition of 14 ml (10 mg/ml finalconcentration) of lysozyme at 100 mg/ml in 25 mM Na acetate, 50%glycerol.

The suspension is centrifuged at 30 000 g for 30 minutes (pellet P2containing the protein; versus supernatant S2 containing thecontaminants of rTbpB). The pellet containing the membranes can befrozen at this stage.

Washing of Membrane Fragments

The lysis pellet P2 is taken up in 50 mM Tris HCl (1100 ml). Afterhomogenization, (Turrax 15 seconds), it is washed for one hour at +4° C.A centrifugation is carried out as previously at 30 000 g for 30minutes. The pellet (P3; versus supernatant S3) is frozen at −45° C. 50mM Tris HCl buffer makes it possible to remove a small amount of protein(supernatant S3) and solubilizes only very little rTbpB.

The pellet P3 is taken up in 50 mM Tris HCl buffer containing 8 M urea,pH 8.0 (800 ml). This buffer makes it possible to remove a part of thecontaminating proteins without solubilizing the membranes containing therTbpB. After homogenization (without using a Turrax), the solution isthen stirred for one hour at +4° C. A centrifugation is carried out aspreviously at 30 000 g for 30 minutes, which makes it possible to obtaina membrane pellet which can be frozen.

Membrane Solubilization

The thawed membrane pellet is solubilized with 780 ml of 50 mM Tris HClbuffer containing 6 mM EDTA, 2 M urea and 4% elugent, at pH 7.5. Thepresence of the detergent at 4% and of the 2 M urea makes it possible tosolubilize the pellet. The solution is stirred at +4° C. overnight(minimum 16 h). Centrifugation of the solution at 30 000 g (1 hour at+4° C.) leaves only a small pellet (P4) containing a few impurities. Thesupernatant S4 containing the rTbpB protein is recovered for loading ona first cation exchange column (QS I).

Purification by Anion Exchange Chromatography on Q Sepharose at pH 7.5

Two successive chromatographies are carried out. The product of thefirst chromatography is collected and then subsequently loaded, after adialysis step, on a second chromatography column which uses differentconditions (absence of EDTA).

1^(st) Chromatography, in the Presence of EDTA (chromatography QS I)

A column of 600 ml (K50, diameter 20 cm²) of Q Sepharose Fast Flow gel(ref 17-0510-01 GE Healthcare) is mounted, tamped in equilibrationbuffer, 50 mM Tris HCl containing 6 mM EDTA, 2 M urea and 1% elugent, atpH 7.5, at the flow rate of 8 ml/minute.

The supernatant S4 (approximately 845 ml) is loaded at the flow rate of6 ml/minute. The direct eluate (part which does not attach to the columnduring loading of the sample) contains the protein of interest, rTbpB.This eluate (1150 ml) is taken and then dialyzed at +4° C. (for 6 days)against 6 liters of 50 mM Tris HCl buffer containing 2 M urea and 1%elugent, pH 7.5, in order to reduce the EDTA concentration to 1 mM andto remove NaCl.

2^(nd) Chromatography (QS II), Without EDTA

A K50 column of 490 ml of new Q Sepharose Fast Flow gel is equilibratedin 50 mM Tris HCl buffer containing 2 M urea and 1% elugent, pH 7.5.

The dialyzed solution (1080 ml) is loaded on the column (flow rate 6ml/minute); then 5 saline elution steps in this same buffer are carriedout: 20 mM, 50 mM, 100 mM, 250 mM and 1 M NaCl (working flow rate 6ml/minute). The rTbpB protein is eluted from the column at two saltconcentrations (50 mM and 100 mM). The 50 mM elution fraction is thefraction of interest, since the rTbpB protein therein is the purest andis present in a greater amount (2.6 times more protein than in the 100mM NaCl fraction).

The pH of the fraction corresponding to the 50 mM NaCl elution peak isdecreased, with magnetic stirring, to pH 5.5 by adding 1.7N acetic acid.The solution (860 ml) is dialyzed against 5 liters of 10 mM sodiumacetate buffer containing 1 M urea and 0.2% elugent, pH 5.5 (24 hours at+4° C.) and then against 4 liters of 10 mM sodium acetate buffercontaining 1 M urea and 0.2% elugent, pH 5.5 (17 hours at +4° C.).

Purification by Cation Exchange Chromatography on SP Sepharose (SPI) atpH 5.5

A K50 column or 100 ml of new SP Sepharose Fast Flow gel (Ge Healthcare,ref 17-0729-01) is equilibrated in 10 mM sodium acetate buffercontaining 1 M urea and 0.2% elugent, pH 5.5.

The dialyzed protein solution (850 ml) is loaded on the column (flowrate 6 ml/minute). Then, five saline elution steps are carried out: 50mM, 100 mM, 250 mM, 500 mM and 1 M NaCl, in the buffer mentioned above.

The rTbpB protein is eluted exclusively in the 250 mM NaCl fraction andthe low-molecular-weight contaminants are eliminated essentially in thedirect eluate (40%).

About 35 mg of purified rTbpB M982 are thus obtained and slightly lessfor rTbpB B16B6.

Dialysis and Concentration of the SPI Product (250 mM fractions)

The fractions corresponding to the 250 mM elution peak of the SPI columnare combined (volume 274 ml). The pH of the solution is brought back upto pH 7.3 by adding, with stirring, approximately 800 μl of 0.5 N NaOH.The solution is dialyzed at +4° C. (Spectra Por 1: cutoff threshold6-8000 D) against two 10 liter baths of PBS containing 0.2% elugent, pH7.1 (66 hours and 22 hours).

The dialysate is concentrated to a volume of 21.1 ml by frontaldiafiltration concentration on a 30 kD Amicon membrane in PBS (ref.PBTK06510).

The concentrate obtained is then again dialyzed against 2 liters of PBScontaining 0.2% elugent, pH 7.1 (Slide A Lyser ref. 66810: cutoffthreshold 10 kD).

The solution is then filtered aseptically through a 0.22 μm Millexfilter with Durapore membrane (Millipore ref. SLGV 033RS). The purifiedrTbpB protein batch obtained is frozen at −80° C. The proteinconcentration is 1642 μg/ml.

1.3. Preparation of rTbpB for Injection

The rTbpB solution obtained in section B.1.2. is treated by adsorptionon Bio-Beads™ SM-2 in order to remove the excess Elugent™ detergent(surfactant in particular constituted of alkyl glucosides) which coulddestabilize the LOS liposomes.

Activation of Bio-Beads™

About 2.5 ml of methanol are added to 500 mg of Bio-Beads™ and themixture is homogenized intermittently for 15 min at ambient temperature.After a settling-out period, the supernatant is removed. This washingoperation is repeated twice.

About 5 ml of ultra-filtered sterile water are then added and themixture is homogenized intermittently for 15 min at ambient temperature.After a settling-out period, the supernatant is removed. This washingoperation is repeated twice.

About 5 ml of PBS are then added and the mixture is homogenizedintermittently for 15 min at ambient temperature. It is stored at 5° C.and used the same day.

At the end, the weight of the Bio-Beads™ has increased by a factor R(equal to approximately 1.2).

Removal of the Detergent by Adsorption on Bio-Beads™

The rTbpB solution obtained in section 1.2. contains 2 mg/ml ofElugent™. The amount of Bio-Beads™ that has to be used is determinedaccording to the amount of Elugent™ to be removed.

For one ml of the rTbpB solution obtained in section B.1.2., 29×R mg ofactivated Bio-Beads™ are added. The mixture is vigorously stirred forone hour at ambient temperature. The maximum amount of liquid is thenrecovered and a final concentration of 0.001% of merthiolate is addedthereto. The whole process is carried out under sterile conditions.

2. Preparation of the Purified LOS Culture

Eight ml of frozen sample of any one of N. meningitidis C708 serogroup Astrains described hereinabove or N. meningitidis strain A1 serogroup Athat exclusively expresses immunotype L8 and exhibit a LOS bearing twoPEAs, one in position 3, one in position 6 of heptose II, are used toinoculate 800 ml of Mueller-Hinton medium (Merck) supplemented with 4 mlof a solution of glucose at 500 g/l and divided up in Erlenmeyer flasks.The culture is continued with shaking at 36±1° C. for approximately 10hours.

400 ml of a solution of glucose at 500 g/l and 800 ml of a solution ofamino acids are added to the preculture. This preparation is used toinoculate a fermentor containing Mueller-Hinton medium, at an OD_(600nm)close to 0.05. The fermentation is continued at 36° C., at pH 6.8, 100rpm, pO₂ 30% under an initial airstream of 0.75 l/min/l of culture.

After approximately 7 hours (OD_(600nm) of approximately 3),Mueller-Hinton medium is added at a rate of 440 g/h. When the glucoseconcentration is less than 5 g/l, the fermentation is stopped. The finalOD_(600nm) is commonly between 20 and 40. The cells are harvested bycentrifugation and the pellets are frozen at −35° C.

Purification (Method Adapted by Westphal & Jann, (1965) Meth. Carbohydr.Chem. 5: 83)

The pellets are thawed and suspended with 3 volumes of 4.5% (vol./vol.)phenol with vigorous stirring for 4 hours at approximately 5° C. The LOSis extracted by phenol treatment.

The bacterial suspension is heated to 65° C. and then mixed vol./vol.with 90% phenol, with vigorous stirring for 50-70 min at 65° C. Thesuspension is subsequently cooled to ambient temperature and thencentrifuged for 20 min at 11 000 g. The aqueous phase is removed andstored, while the phenolic phase and the interphase are harvested so asto be subjected to a second extraction.

The phenolic phase and the interphase are heated to 65° C. and thenmixed with a volume of water equivalent to that of the aqueous phasepreviously removed, with vigorous stirring for 50-70 min at 65° C. Thesuspension is subsequently cooled to ambient temperature and thencentrifuged for 20 min at 11 000 g. The aqueous phase is removed andstored, while the phenolic phase and the interphase are harvested so asto be subjected to a third extraction identical to the second.

The three aqueous phases are dialyzed separately, each against 401 ofwater. The dialysates are then combined. One volume of 20 mM Tris, 2 mMMgCl₂ is added to 9 volumes of dialysate. The pH is adjusted to 8.0±0.2with 4 N sodium hydroxide.

Two hundred and fifty international units of DNAse are added per gram ofpellet. The pH is adjusted to 6.8±0.2. The preparation is placed at 37°C. for approximately 2 hours with magnetic stirring, and then subjectedto filtration through a 0.22 μm membrane. The filtrate is purified bypassing it through a Sephacryl S-300 column (5.0×90 cm; Pharmacia™).

The fractions containing the LOS are combined and the MgCl₂concentration is increased to 0.5 M by adding powdered MgCl₂.6H₂O, withstirring.

While continuing the stirring, dehydrated absolute alcohol is added togive a final concentration of 55% (vol./vol.). The stirring is continuedovernight at 5±2° C., and then centrifugation is carried out at 5000 gfor 30 min at 5±2° C. The pellets are resuspended with at least 100 mlof 0.5 M MgCl₂ and then subjected to a second alcoholic precipitationidentical to the preceding one. The pellets are resuspended with atleast 100 ml of 0.5 M MgCl₂.

The suspension is subjected to a gel filtration as previously described.The fractions containing the LOS are combined and filtration-sterilized(0.8-0.22 μm) and stored at 5±2° C.

This purification method makes it possible to obtain approximately 150mg of LOS per liter of culture.

3. Preparation of [LOS] Liposomes by Detergent Dialysis 3.1. Preparationof Liposomes

The LOS liposomes are prepared by detergent dialysis. Briefly, thelipids (EDOPC:DOPE) are made into the form of a lipid film and taken upin 10 mM Tris buffer, and then dispersed in the presence of 100 mM ofoctyl-β-D-glucopyranoside (OG) (Sigma-Aldrich ref. 08001) and filteredsterilely. The LOS in 100 mM OG is added sterilely. The lipids/LOS/OGmixture is then dialyzed against 10 mM Tris buffer in order to removethe OG and to form the liposomes.

Protocol

A lipid preparation in chloroform, of the lipids that will be used toproduce the liposomes, is prepared. A dry film is obtained by completeevaporation of the chloroform.

A dry film of 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC orethyl-DOPC) and of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)in an EDOPC:DOPE mole ratio of 3 to 2 is obtained by mixing 12.633 ml ofa solution of EDOPC (Avanti Polar Lipids ref. 890704) at 20 mg/ml inchloroform and 7.367 ml of a solution of DOPE (Avanti Polar Lipids ref.850725) at 20 mg/ml in chloroform, and evaporating off the chloroformuntil it has completely disappeared.

The dry film is taken up with 30 ml of 10 mM Tris buffer, pH 7.0, so asto obtain a suspension containing 13.333 mg of lipids/ml (8.42 mg/ml ofEDOPC and 4.91 mg/ml of DOPE). The suspension is stirred for 1 hour atambient temperature and then sonicated for 5 min in a bath.

3.333 ml of a sterile 1 M solution of octyl-β-D-glucopyranoside (OG)(Sigma-Aldrich ref. 08001) in 10 mM Tris buffer, pH 7.0, are then added,still with stirring, so as to obtain a clear suspension of lipids at 12mg/ml, 100 mM OG and 10 mM Tris buffer. The stirring is continued for 1h at ambient temperature on a platform shaker. Filtration is thencarried out sterilely through a Millex HV 0.45 μm filter.

A composition is prepared, under sterile conditions, by bringingtogether LOS and lipids in a lipids:LOS mole ratio of 250 (0.160 mg/mlof LOS, 9.412 mg/ml of lipids and 100 mM of OG). 40 ml of such acomposition are obtained from mixing the following preparations:

2.005 ml of 10 mM Tris buffer, pH 7.0; 0.223 ml of 100 mM OG in 10 mMTris; 31.373 ml of the EDOPC:DOPE suspension having a mole ratio of 3:2,at 12 mg/ml in 100 mM OG, 10 mM Tris; and 6.4 ml of a sterile suspensionof LOS at 1 mg/ml in 100 mM OG, 10 mM Tris.

After stirring for one hour at ambient temperature, the suspension istransferred sterilely into 4 sterile 10 ml dialysis cassettes. Eachcassette is dialyzed 3 times (24 hrs-24 hrs-72 hrs) against 200 volumesof 10 mM Tris, pH 7.0, i.e. 21.

The liposomes are recovered under sterile conditions. The increase involume after dialysis is approximately 30%.

Merthiolate and NaCl are added to this preparation so as to obtain apreparation of liposomes in 10 mM Tris, 150 mM NaCl, pH 7.0, 0.001%merthiolate, which ultimately contains approximately 110 μg/ml of LOSand 7 mg/ml of lipids, of which there are approximately 4.5 mg/ml ofEDOPC and approximately 2.5 mg/ml of DOPE (theoretical concentrations).

The LOS liposomes are stored at +5° C.

3.2. Preparation of the Injectable Materials

The liposomes are adjusted to the required LOS concentration (inparticular required for the immunogenicity tests) in 10 Mm Tris, 150 MmNaCl, pH 7.4. The merthiolate concentration is maintained at 0.001%.

4. Preparation of an [LOS] Liposomes +rTbpB Mixture

rTbpB in PBS (section B.1.3.) is mixed with [LOS] liposomes (sectionB.3.) in an rTbpB:LOS weight:weight ratio equal to 1. The volume is thenadjusted with 10 mM Tris buffer containing 150 mM NaCl, pH 7.4, so as toobtain a preparation in which each of the components (rTbpB and LOS) isat a concentration of 80 μg/ml. The merthiolate concentration ismaintained at 0.001%.

5. Production of an Endotoxoid (LOS Detoxified by Complexation with aPeptide Analog of Polymyxin B)

This endotoxoid is prepared as described in patent application WO06/108586. Briefly, one volume of a solution of purified LOS at 1 mg/ml,sterilized by filtration through a 0.22 μm membrane, is mixed with onevolume of a solution of SAEP2-L2 peptide at 1 mg/ml, sterilized byfiltration through a 0.22 μm membrane.

The SAEP2-L2 peptide (SEQ ID NO: 3) is a peptide with an antiparalleldimeric structure, of formula:

A precipitate forms immediately. Mixing is carried out for 5 min atambient temperature, and then the mixture is left to stand overnight at4° C. The precipitate is harvested by centrifugation at 3000 rpm for 10min. The pellet is washed 5 times with one volume of pyrogen-freesterile water, pH 7.2. Finally, the pellet is resuspended in 10 mM Trisbuffer containing 150 mM NaCl and Tween 80, pH 7.4, so as to obtain asuspension at 1 mg/ml, calculated based on the wet weight of theprecipitate. The suspension is stored at 4° C.

6. Preparation of an Endotoxoid +rTbpB Mixture

rTbpB in PBS (obtained as described in section B.1.3.) is mixed withendotoxoid (section B.5.) in a weight:weight ratio equal to 1. Thevolume is then adjusted with 10 mM Tris buffer containing 150 mM NaCland 0.05% Tween 80 so as to obtain a preparation in which each of thecomponents is at a concentration of 80 μg/ml.

7. Immunogenicity Study No. 1 in Rabbits

The various formulations tested were produced as described in one of thepreceding sections.

7.1. Immunization of Rabbits

Twenty-four 7-week-old female NZ-KBL rabbits (Charles River Lab.) aredivided up into 5 test groups of four and 2 control groups of two.

The female rabbits of each group receive, in a volume of 0.5 ml, dividedup into 2 concomitant intramuscular injections in the legs, at DO, D21and D42:

-   Group A: 40 μg of liposomes [LOS α chain L6, PEA-3] in 10 mM Tris,    150 mM NaCl, pH 7.4 buffer;-   Group B: 40 μg of liposomes [LOS α chain L6, PEA-3] and 40 μg M982    rTbpB in 10 mM Tris, 150 mM NaCl, pH 7.4 buffer;-   Group C: 40 μg of liposomes [LOS α chain L6, PEA-3, PEA-6] in 10 mM    Tris, 150 mM NaCl, pH 7.4 buffer;-   Group D: 40 μg of liposomes [LOS α chain L6, PEA-3, PEA-6] and 40 μg    M982 rTbpB in 10 mM Tris, 150 mM NaCl, pH 7.4 buffer;-   Group E: 40 μg of LOS α chain L6, PEA-3 in endotoxoid form and 40 μg    M982 rTbpB in 10 mM Tris, 150 mM NaCl, 0.5% Tween, pH 7.0 buffer;-   Group F (control): 40 μg rTbpB and empty liposomes in 10 mM Tris,    150 mM NaCl, pH 7.4 buffer; and-   Group G (control): empty liposomes in 10 mM Tris, 150 mM NaCl, pH    7.4 buffer.

A blood sample is taken from the animals for analysis at DO, D42 (beforethe third injection) and at D56.

7.2. Assaying of Anti-LOS Antibodies by ELISA

This assay is automated (Staccato automation system, Caliper) accordingto the following protocol:

The wells of Dynex™ 96-well plates are impregnated with, for each of thegroups, 1 μg of LOS homolog in 1×PBS (phosphate buffered saline) buffer,pH 7.1, 10 mM MgCl₂, and the plates are incubated for 2 hours at 37° C.and then overnight at 4° C. The plates are blocked by adding, to thewells, 150 μl of PBS containing 0.05% of Tween 20 and 1% (weight/vol) ofskimmed milk powder (PBS-Tween-milk). The plates are incubated for 1hour at 37° C.

Serial doubling dilutions of the test samples are prepared in PBS—0.05%Tween—1% milk. The plates are incubated for 90 min at 37° C. and thenwashed 3 times with PBS +Tween 20 at 0.05%.

A peroxidase-anti-mouse IgG or peroxidase-anti-rabbit IgG conjugate inPBS-Tween-milk is added to the wells and the plates are incubated for 90min at 37° C. The plates are washed three times. 100 μl of aready-to-use solution of TMB (3,3′,5,5′-tetramethylbenzidine, substratefor peroxidase) are distributed per well. The plates are incubated inthe dark for 20 min at ambient temperature. The reaction is stopped byadding 100 μl of 1 M HCl per well.

The optical density is measured at 450-650 nm with an automatic reader(Multiskan Ascent). In the absence of standard, the antibody titers aredetermined as being the reciprocal dilution giving an optical density of1.0 on a tendency curve (CodUnit software). The antibody detectionthreshold is 1.3 log₁₀ ELISA unit. For each titer below this threshold,an arbitrary value of 1.3 log₁₀ is assigned.

7.3. Measurement of the Bactericidal Activity of IgGs Purified AgainstN. meningitidis Strains Heterologous to the Strain C708 (SBA test)

IgGs were purified from pooled sera by affinity chromatography usingHiTrap rProtein A FF column (GE Healthcare/Amersham Biosciences)according to the supplier's recommendations.

On the basis of the purified IgGs, serial twofold dilutions are carriedout in gelatin-containing Dulbecco's PBS with calcium and magnesiumions. The dilutions are carried out in a 96-well plate for a finalvolume of 50 μl per well.

The bactericidal activity of the purified IgGs has been tested againstthe strains mentioned in the table which follows:

Twenty-five μl of a culture of N. meningitidis in the exponential phase(4×10³ CFU/ml) in BHI medium in the absence of an agent which chelatesiron in free form (so as to avoid expressing TbpB), and also 25 μl ofbaby rabbit complement at 1/1.5, are added to each well. The plate isincubated for one hour at 37° C., with shaking.

Fifty μl of the mixture of each well are then deposited on bioMérieuxMueller-Hinton agar plates and incubated overnight at 37° C. under 10%CO₂. The number of clones is counted.

There are three controls:

-   bacteria+baby rabbit complement, without test serum (“complement”    control);-   bacteria+inactivated baby rabbit complement, without test serum    (“microorganism” control); and-   bacteria+inactivated baby rabbit complement +test serum (serum    control).

The bactericidal titer is expressed as the inverse of the dilutiongiving 50% bacterial death by comparison with the “complement” control.

7.4. Results and Discussion ELISAs

FIG. 3 gives the ELISA titers expressed as log₁₀ of the anti-LOS IgGs ofthe rabbit sera of groups A, B, C, D, E and F. In white, the titers ofthe sera before immunization; shaded, those of the sera of which sampleswere taken at D42 after the second immunization; and darkly shaded,those of the sera of which samples were taken at D56.

The ELISA titers at times D42 and D56 show that the LOS obtained fromeach of the strains C708 constructed as described previously isimmunogenic. This immunogenicity is increased by the presence oflipidated rTbpB for each period under consideration (which confirms theadjuvant power of this lipoprotein).

SBA Test

The bactericidal activity has been established in “fold increase” asbeing the purified IgG bactericidal titer of a considered group:negative control group titer ratio. As such, the seroconversion rate in“fold increase” expresses the bactericidal titer increase. It isconsidered that the bactericidal activity is significant when a “foldincrease” equal or above ×8 is obtained.

Purified IgGs from control group G exhibit against all tested strains a

fold increase

inferior to ×4.

The

fold increase

values obtained with purified IgGs from groups B and D against 18strains are reported in table IV hereinafter:

Purified IgGs obtained after immunisation with the group B composition(40 liposomes [LOS chaine a L6, PEA-3] and 40 μg rTbpB M982) have thenbeen tested against a larger number of strains. The results are reportedin tables V and VI hereinafter.

8. Immunogenicity Study No. 2 in Rabbits

The various test formulations were manufactured as described in one ofthe preceding sections.

8.1. Immunization of Rabbits

Twenty-four 7-week-old NZ KBL female rabbits (Charles River Lab.) aredispatched into 4 test groups of four (groups A to D) and 4 groups of 2(groups E and H).

The female rabbits of each group receive in a volume of 0.5 ml dividedamong 2 concomitant intramuscular injections into the legs, on DO, D21and D42:

-   Group A: 40 μg of liposomes [LOS α chain L8, PEA-3, PEA-6] et 40 μg    rTbpB M982, in 10 mM Tris, 150 mM NaCl, pH 7.4 buffer;-   Group B: 40 μg of liposomes [LOS α chain L8, PEA-3, PEA-6] and 40 μg    rTbpB B16B6, in 10 mM Tris, 150 mM NaCl, pH 7.4 buffer;-   Group C: 40 μg of liposomes [LOS α chain L8, PEA-3] and 40 μg rTbpB    M982, in 10 mM Tris, 150 mM NaCl, pH 7.4 buffer;-   Group D: 40 μg of liposomes [LOS α chain L8, PEA-3, PEA-6] in 10 mM    Tris, 150 mM NaCl, pH 7.4 buffer;-   Group E: 40 μg rTbpB M982 and 40 μg of LOS-free liposomes in 10 mM    Tris, 150 mM NaCl, 0.5% Tween, pH 7.0 buffer;-   Group F: 40 μg rTbpB B16B6 and 40 μg of LOS-free liposomes in 10 mM    Tris, 150 mM NaCl, 0.5% Tween, pH 7.0 buffer;-   Group G: 40 μg of liposomes [LOS α chain L8, PEA-3] in 10 mM Tris,    150 mM NaCl, pH 7.4 buffer;-   Group H: 10 mM Tris, 150 mM NaCl, pH 7.4 buffer

Blood is collected from the animals for analysis at DO, D42 (before thethird injection) and at D56.

8.2. Measurement of the Bactericidal Activity of Purified IgGs AgainstStrains of N. meningitidis Heterologous to the Strain C708

IgGs were purified from pooled sera by affinity chromatography using theHiTrap rProtein A FF column (GE Healthcare/Amersham Biosciences)according to the manufacturer's recommendations.

Using the purified IgGs, twofold serial dilutions are performed inDulbecco's gelatinized PBS containing calcium and magnesium ions. Thedilutions are performed in a 96-well plate for a final volume of 50 μlper well.

The bactericidal activity of the purified IgGs was tested against thestrains mentioned in Table V below:

25 μl of an N. meningitidis culture in the exponential phase (4×10³CFU/ml) in BHI medium +50 μM of Desferal (agent for chelating iron infree form, to allow the expression of TbpB) and 25 μl of baby rabbitcomplement at 1/1.5 are added to each well. The plate is incubated forone hour at 37° C. with stirring.

50 μl of the mixture in each well are then deposited on bioMérieuxMueller-Hinton agar dishes and incubated overnight at 37° C. under 10%CO₂. The number of clones is counted.

There are three controls:

-   bacteria +baby rabbit complement, without test serum (“complement”    control);-   bacteria +inactivated baby rabbit complement, without test serum    (“microorganism” control); and-   bacteria +inactivated baby rabbit complement +test serum (serum    control).

The bactericidal titer is expressed as being the inverse of the dilutiongiving 50% bacterial death by comparison with the “complement” control.

8.3. Results and Discussion

SBA test

34 N. meningitidis strains have been tested for cross bactericidalactivity. Their names are to be seen in table V hereinafter. The resultsare expressed in

fold increase

according to the calculation methodology described in section B.7.4.hereinabove.

As expected, purified IgGs from negative control immunisation group donot show any bactericidal activity against any of the strains.

Purified IgGs from immunisation groups D and G (LOS not adjuvanted withTbpB) exhibit a bactericidal activity of less interest. For simplicity'sake, table V hereinafter only shows the SBA results expressed in

fold increase

, obtained with purified IgGs from groups A, B, C, E and F of the secondstudy together with the results obtained with the purified IgGs of groupB of the first study.

Table VI shows the SBA results expressed in

fold increase⊖, of purified IgGs from immunisation group B of the firststudy and of group A of the second study against 22 strains cultured inpresence/absence of Desferal.

Table VII shows for a variety of vaccine compositions, the percentage ofprotection deduced from the cross SBA studies including 34 strainscultured in presence of Desferal.

TABLE VII % of protection deduced from cross SBA studies including 34strains cultured in the Vaccine composition presence of Desferal L6 PEAO3 + TbpB M982 61.8% L8 PEA O3, O6 + TbpB M982 55.9% L6 PEA O3 + L8 PEAO3, O6 + TbpB M982 70.6% L8 PEA O3 + TbpB M982 52.9% L6 PEA O3 + L8 PEAO3 + TbpB M982 67.6% L8 PEA O3, O6 + TbpB B16B6   32% L6 PEA O3 + TbpBM982 + TbpB B16B6 73.5% L8 PEA O3, O6 + TbpB M982 + TbpB B16B658.8-67.6% L6 PEA O3 + L8 PEA O3, O6 + 82.4% TbpB M982 + TbpB B16B6 L8PEA O3 + TbpB M982 + TbpB B16B6 64.7%

1. A process of making a modified Neisseria meningitidis strainexhibiting a lipooligosaccharide (LOS) comprising a lipid A, an innercore, and an L8 type α chain in which the heptose II residue of theinner core bears in position O-3 and in position O-6 or O-7 aphosphoethanolamine (PEA) substituent, wherein the process comprisesmodifying an N. meningitidis strain of immunotype L8 so that itexpresses an N. meningitidis lpt6 gene.
 2. The process according toclaim 1 wherein the Neisseria meningitidis strain that is modified is ofserogroup A.
 3. A modified Neisseria meningitidis strain made accordingto the process of claim
 1. 4. A modified Neisseria meningitidis strainmade according to the process of claim
 2. 5. A process for preparing alipooligosaccharide (LOS) of N. meningitidis, the process comprising (a)culturing a modified Neisseria meningitidis strain of claim 3; and (b)harvesting the LOS from the culture of (a); wherein the LOS comprises alipid A, an inner core, and an α chain of L8 type, and wherein theheptose II residue of the inner core bears in position O-3 and inposition O-6 or O-7 a phosphoethanolamine (PEA) substituent.
 6. Aprocess for preparing a lipooligosaccharide (LOS) of N. meningitidis,the process comprising (c) culturing a modified Neisseria meningitidisstrain of claim 4; and (d) harvesting the LOS from the culture of (a);wherein the LOS comprises a lipid A, an inner core, and an α chain of L8type, and wherein the heptose II residue of the inner core bears inposition O-3 and in position O-6 or O-7 a phosphoethanolamine (PEA)substituent.
 7. The process according to claim 5 wherein the LOS isharvested in a form combined with outer membrane vesicles (OMVs).
 8. Theprocess according to claim 5 wherein the LOS is extracted for furtherpurification.
 9. The process according to claim 6 wherein the LOS isharvested in a form combined with outer membrane vesicles (OMVs). 10.The process according to claim 6 wherein the LOS is extracted forfurther purification.
 11. A lipooligosaccharide (LOS) of N. meningitidisprepared by the process of claim
 5. 12. A lipooligosaccharide (LOS) ofN. meningitidis prepared by the process of claim
 6. 13. Alipooligosaccharide (LOS) of N. meningitidis prepared by the process ofclaim
 7. 14. A lipooligosaccharide (LOS) of N. meningitidis prepared bythe process of claim
 8. 15. A lipooligosaccharide (LOS) of N.meningitidis prepared by the process of claim
 9. 16. Alipooligosaccharide (LOS) of N. meningitidis prepared by the process ofclaim 10.